Viral proteins capable of binding LAR

ABSTRACT

A poxvirus protein designated A41L binds to leukocyte common-antigen-related protein (LAR). A41L is a secreted protein that can be purified from the culture supernatant of cells infected with certain poxviruses, or produced using recombinant DNA techniques. A41L polypeptides and LAR polypeptides, and nucleic acids encoding them, are provided herein. Also provided are methods of using such polypeptides and nucleic acids.

This application is a divisional of U.S. application Ser. No. 10/191,029filed Jul. 3, 2002, which claims the benefit of U.S. provisionalapplication Ser. No. 60/305,223 filed Jul. 12, 2001, both of which areincorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

Poxviruses form a group of large, double-stranded DNA viruses that haveadapted to replicate in numerous hosts. One adaptive mechanism that manypoxviruses have utilized is the acquisition of host genes that allow theviruses to evade the host's immune system and/or facilitate viralreplication (Bugert and Darai, Virus Genes 21:111, 2000; Alcami et al.,Semin. Virol. 8:419, 1998; McFadden and Barry, Semin. Virol. 8:429,1998). This process has been facilitated by the relatively large sizeand complexity of the poxvirus genome; vaccinia virus, a prototypepoxvirus widely used as a smallpox vaccine, has a genome ofapproximately 190 Kbp that could potentially encode more than 200proteins (Goebel et al., Virology 179:247, 1990). Despite the fact thatthe entire genome of vaccinia virus has been sequenced, the function ofmany of the potential open reading frames (ORFs), and the existence ofpolypeptides encoded thereby, remains unknown.

A41L is an ORF present in several different poxviruses, including Cowpoxvirus (CPV), vaccinia virus (strains Copenhagen, Ankara, Tian Tan andWR) and variola virus (including strains Harvey, India-1967 andGarcia-1966). An ORF from vaccinia strain WR referred to as SalF4L inHoward et al., Virology 180:633, 1991, was noted to exhibit weeksimilarity with an ORF from Shope fibroma virus (SFV) referred totherein as T1. The protein encoded by the SFV T1 ORF is referred to asp35 in Martinez-Pomarres et al. (Virology 206:591, 1995), who state thatSalF4L did not exhibit significant homology with p35. p35 binds to, andinhibits the activity of, certain chemokines (Smith et al., Virology236:316, 1997; U.S. Pat. No. 5,871,740). An A41L protein made byrecombinant baculovirus expression was reported to specifically bind thechemokines Mig and IP-10, but not other chemokines, by Smith et al. inWO98/37217.

The leukocyte common-antigen-related protein (LAR) is a prototypicmember of the superfamily of receptor-like protein tyrosine phosphatases(PTPs) with immunoglobulin and fibronectin type III-like motifs in theextracellular domain (Streuli et al., J. Exp. Med. 168:1523, 1988).Several alternatively spliced variants of LAR have been identified, andare believed to be developmentally regulated (O'Grady et al., J. Biol.Chem. 269:25193, 1994; Zhang and Longo, J. Cell. Biol. 128:415, 1995;Honkaniemi et al., Mol. Brain. Res. 61:1, 1998). In humans, the LAR genemaps to chromosome 1p32, a region that is frequently deleted in tumorsof neuroectodermal origin (Jirik et al., Cytogenet. Cell Genet. 61:266,1992).

Changes in LAR expression and splicing have been associated with changesin the ability of cells to proliferate (Yang et al., Carcinogenesis21:125; Tisi et al., J. Neurobiol. 42:477, 2000). Transfection of ahuman breast carcinoma cell line that overexpresses the protein tyrosinekinase p185^(neu) with cDNA for LAR resulted in suppression of tumorcell growth (Zhai et al., Mol. Carcinogen. 14:103, 1995), suggesting arole for LAR as a tumor suppressor. The related PTP, CD45, suppressesJanus kinase (JAK) kinases and negatively regulates cytokine receptorsignaling; LAR also dephosphorylates JAK2 (Irie-Sasaki et al., Nature409:349, 2001). LAR has also been found to associate with the insulinreceptor, and play a role in glucose homeostasis (Ahmad and Goldstein,J. Biol. Chem. 272:448, 1997; Ren et al., Diabetes 47:493, 1998). Theseand other functional roles of LAR are discussed in EP 1 092 772(Yamamoto et al., 2001).

Heretofore, the function of any peptide encoded by an A41L ORF wasunclear. Moreover, despite a role being known for LAR in glucosemetabolism and/or cell replication, it was previously unknown whetherLAR plays a role in an immune or inflammatory response, or what suchrole might be. Accordingly, there is a need in the art to determine thebiologic function(s) of a protein or proteins encoded by an A41L ORF,and to determine the role of LAR in an immune or inflammatory response.

SUMMARY OF THE INVENTION

The present invention provides a protein designated A41L that is capableof binding to leukocyte common-antigen-related protein (LAR), a proteintyrosine phosphatase (PTPase). A method for treating a disorder mediatedby LAR involves administering an effective amount of a LAR agonist orantagonist to a mammal afflicted with such a disorder. Preferably thebiological activity of LAR that is regulated is its ability todephosphorylate specific proteins (for example, JAKs).

Isolated nucleic acids encoding A41L are also provided herein, alongwith expression vectors comprising the A41L DNA. Methods for producingrecombinant A41L polypeptides involve culturing host cells containingthe expression vectors under conditions appropriate for expression ofA41L, and recovering the expressed A41L from the cell culture. Certainembodiments of the invention are directed to A41L DNA derived fromcowpox virus, the A41L protein encoded thereby, and uses thereof.

The present invention also provides methods for screening for a moleculethat alters (antagonizes or agonizes) an activity of LAR. In one aspect,the inventive methods utilize homogeneous assay formats such asfluorescence resonance energy transfer, fluorescence polarization,time-resolved fluorescence resonance energy transfer, scintillationproximity assays, reporter gene assays, fluorescence quenched enzymesubstrate, chromogenic enzyme substrate and electrochemiluminescence. Inanother aspect, the inventive methods utilize heterogeneous assayformats such as enzyme-linked immunosorbant assays (ELISA) orradioimmunoassays. In yet another aspect of the invention are cell-basedassays, for example those utilizing reporter genes, as well asfunctional assays that analyze the effect of an antagonist or agonist onbiological function(s).

The invention further provides methods for producing informationcomprising the identity of a compound that alters one or more biologicalactivities of LAR, the method comprising using assay methods of theinvention to identify one or more compounds that alter the binding ofA41L and LAR. In one preferred embodiment, the compound decreases (orantagonizes) the binding of A41L and LAR, and in another distinctembodiment, the compound increases (or agonizes) the binding of A41L andLAR.

Also provided by the invention is the information produced according tothe inventive methods, said information comprising the identity of acompound that alters the biological activity of LAR, and preferablyembodied in a storage medium selected from the group consisting ofpaper, magnetic tape, optical tape, floppy disks, compact disks,computer system hard drives, and computer memory units. In a furtheraspect, the invention provides a database comprising said information,wherein the information is preferably embodied in a computer-readablemedium, and a separate embodiment wherein the information is embodied ina human-readable medium.

Additionally provided by the invention is a computer system comprising adatabase containing records pertaining to a plurality of compounds,wherein the records comprise results of an assay of the invention, and auser interface allowing a user to access information regarding theplurality of compounds. In another aspect of the invention, a computersystem is provided for storing and retrieving data on a plurality ofcompounds, the computer system comprising: (a) input means for enteringdata for the compounds into a storage medium; (b) a processor forcreating an individual record for each compound, the processor assigningspecific identifying values for each compound; (c) means for selectingone or more of the records based on results in an assay; and (d) meansfor transmitting information in the record or records to an outputdevice to produce a report; preferably a report in human-readable form,and wherein the computer system preferably further comprises a videodisplay unit.

The invention also provides a method of using the computer system of theinvention to select one or more compounds for testing from a pluralityof compounds having records stored in a database, the method comprising:displaying a list of said records or a field for entering informationidentifying one or more of said records; and selecting one or more ofthe records from the list or the record or records identified byentering information in the field. Further, the invention provides amethod of operating a computer system for analyzing compounds thatmodulate the interaction of A41L and LAR, the method comprising: (a)entering data relating to a plurality of compounds into a storagemedium; (b) processing the data to create an individual record for eachcompound; (c) testing compounds for the ability to modulate binding ofA41L to LAR; and (d) communicating results from the testing into thestorage medium such that results for each compound are associated withthe individual record for that compound; wherein in one embodiment thestorage medium comprises one or more computer memory units, and inanother embodiment the computer system further comprises a video displayunit.

In yet another aspect of the invention, a database is providedcomprising records generated according to the methods of the invention,and a method is provided for selecting compounds that modulate theinteraction of A41L and LAR, comprising compiling said database,analyzing the testing results, and selecting one or more compounds.

Candidate molecules that are determined to alter an activity of LAR areuseful, for example, for the further definition of LAR-mediatedsignaling pathways, and for the manipulation of LAR-mediated cellularresponses. Moreover, LAR signaling agonists and antagonists providetherapeutic agents for an immune and/or inflammatory response.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an alignment of several members of the A41L gene family.Conserved cysteines are shown in bold face type.

DETAILED DESCRIPTION OF THE INVENTION

The ability of a protein designated A41L to bind LAR is disclosedherein. A41L is encoded by an open reading frame (ORF) present inseveral different poxviruses. The present invention provides a methodfor modulating a biological activity of LAR by contacting a cellexpressing LAR with an agonist or antagonist of LAR. A disorder mediatedby LAR is treated by administering an effective amount of an agonist orantagonist of LAR to a mammal afflicted with such a disorder.

The present invention provides purified A41L polypeptides andpharmaceutical compositions containing such polypeptides. Isolatednucleic acids encoding A41L are provided herein, along with expressionvectors comprising the A41L nucleic acid. Methods for producingrecombinant A41L polypeptides involve culturing host cells containingthe expression vectors under conditions appropriate for expression ofA41L, and recovering the expressed A41L from the cell culture. Certainembodiments of the invention are directed to A41L nucleic acids andpolypeptides, and uses thereof.

As used herein, A41L polypeptide refers to a group of secreted, solublepolypeptides that are poxvirus virulence factors and encoded in a numberof poxvirus genomes, including, but not limited to, variola, cowpox,vaccinia, myxoma, and Shope fribroma viruses. The A41L polypeptides arecapable of specifically binding known LAR. A number of A41L polypeptidesand their amino acid sequences are shown in FIG. 1. References to A41Lpolypeptides herein additionally encompass variants of A41Lpolypeptides, as described herein.

A DNA and encoded amino acid sequence for cowpox A41L is presented inSEQ ID NO:1 and SEQ ID NO:2, respectively. Another A41L protein suitablefor use in the present invention is encoded by an open reading frame inthe genome of the Copenhagen strain of vaccinia virus. The DNA sequenceof the genome for this strain of vaccinia virus is known; the amino acidsequence of a putative A41L ORF is presented herein in SEQ ID NO:3. Thecowpox A41L amino acid sequence in SEQ ID NO:2 is 93% identical to thevaccinia virus A41L amino acid sequence presented in SEQ ID NO:3.Additional strains of vaccinia virus also include putative ORFscorresponding to A41L; these include strain Tian Tan (SEQ ID NO:4),strain Ankara (SEQ ID NO:5), and strain WR (SEQ ID NO:6; a deleted formof this ORF has also been reported in Blasco et al. (J. Virol. 65:4598,1991) and is shown in FIG. 1.

Another suitable A41L protein is encoded by an open reading frame in thegenome of several strains of variola (smallpox) virus. A putative A41Lprotein from variola minor virus, strain Garcia-1966 is presented in SEQID NO:7; a putative A41L protein from variola virus, strain India-1967is shown in SEQ ID NO:8; and ; a putative A41L protein from variolamajor virus, strain Harvey is shown in SEQ ID NO:9. The amino acidsequence shown in SEQ ID NO:8 is 90% identical to that of the cowpoxA41L of SEQ ID NO:2.

Homologs of certain viral proteins have been isolated from mammaliancell sources. Thus, the present invention encompasses homologs of theviral A41L proteins, wherein the homologs are derived from higherorganisms, including mammalian cells. Cowpox A41L DNA may beradiolabeled and used as a probe in cross-species hybridizationprocedures, to detect A41L DNAs in the genomes of other virus strains,or in nucleic acids derived from cells of higher organisms.Alternatively, the nucleotide and/or amino acid sequences in variousdatabases can be examined for homology to A41L proteins, and homologousmammalian (or other species) proteins and the DNAs encoding themidentified and isolated.

The above-described A41L proteins comprise a hydrophobic region at theN-terminus of the protein that is believed to function as a signalpeptide. The signal peptide is predicted to be cleaved after amino acid19 of SEQ ID NO:2 (which corresponds to amino acid 20 of SEQ ID NOs:3through 9). Thus, mature A41L proteins include those comprising aminoacids 20 through 218 of SEQ ID NO:2, amino acids 21 through 219 of SEQID NOs:3, 4, 5, and 6, and amino acids 21 through 218 of SEQ ID NOs:7,8, and 9.

Additionally, N-terminal amino acid sequencing of purified CPV A41Ldemonstrated that the N-terminal amino acid residue is the Thr at aminoacid number 17 of SEQ ID NO:2 (which corresponds to amino acid number 18of SEQ ID NOs:3 through 9). Accordingly, mature A41L proteins alsoinclude those comprising amino acids 17 through 218 of SEQ ID NO:2,amino acids 18 through 219 of SEQ ID NOs:3, 4, 5, and 6, and amino acids18 through 218 of SEQ ID NOs:7, 8, and 9.

Regarding the foregoing discussion of signal peptides and mature A41Lprotein, the skilled artisan will recognize that the above-describedboundaries of such regions of the protein are approximate. For example,although computer programs that predict the site of cleavage of a signalpeptide are available, cleavage can occur at sites other than thosepredicted. Further, it is recognized that a protein preparation cancomprise a mixture of protein molecules having different N-terminalamino acids, due to cleavage of the signal peptide at more than onesite. In addition, post-translational processing can vary according tothe particular expression system employed. Thus, the N- or C-terminalamino acid of a mature recombinant protein may vary according to thetype of host cells in which the protein was expressed, for example.

The invention further provides various forms of cowpox A41L proteins,including a protein comprising amino acids x to y of SEQ ID NO:2,wherein x represents an integer from 1 to 25, inclusive, and yrepresents an integer from 208 to 218, inclusive. Also provided arevarious forms of vaccinia virus A41L proteins, including a proteincomprising amino acids x to y of SEQ ID NO:3, 4, 5 or 6, wherein xrepresents an integer from 1 to 26, inclusive, and y represents aninteger from 209 to 219, inclusive, of SEQ ID NOs:3, 4, 5, or 6, as wellas various forms of variola virus A41L proteins, including a proteincomprising amino acids x to y of SEQ ID NO:7, 8, or 9, wherein xrepresents an integer from 1 to 26, inclusive, and y represents aninteger from 208 to 218, inclusive, of SEQ ID NOs:7, 8, or 9.

Although a protein encoded by an A41L ORF has not been purified fromvirally infected cells, when the A41L ORF is expressed recombinantly, itis a secreted protein. Accordingly, it is believed that non-recombinantA41L may be purified from the culture supernatant of cells infected withA41L-encoding viruses, as described below. Expression and purificationof recombinant A41L is also discussed further below.

LAR-binding fragments of A41L polypeptides may be employed in thepresent invention. The ability of an A41L fragment (or any other A41Lpolypeptide) to bind LAR can be confirmed using a binding assay such asthose described in examples 3 and 4. A41L fragments may be prepared byany of a number of conventional techniques. A desired DNA sequence maybe chemically synthesized, for example. DNA fragments also may beproduced by restriction endonuclease digestion of a full length clonedDNA sequence, and isolated by electrophoresis on agarose gels. Linkerscontaining restriction endonuclease cleavage site(s) may be employed toinsert the desired DNA fragment into an expression vector, or thefragment may be digested at cleavage sites naturally present therein.The well known polymerase chain reaction (PCR) procedure also may beemployed to isolate a DNA sequence encoding a desired protein fragment.Oligonucleotides that define the termini of the desired fragment areemployed as 5′ and 3′ primers in such a PCR procedure. As a furtheralternative, known mutagenesis techniques may be employed to insert astop codon at a desired point, e.g., immediately downstream of the codonfor the last amino acid of a desired fragment.

The present invention provides purified A41L polypeptides, bothrecombinant and non-recombinant. Variants and derivatives of native A41Lproteins that retain the desired biological activity (e.g., the abilityto bind LAR) are also within the scope of the present invention. A41Lvariants may be obtained by mutations of nucleotide sequences coding fornative A41L polypeptides, for example. An A41L variant, as referred toherein, is a polypeptide substantially homologous to a native A41L, butwhich has an amino acid sequence different from that of a native A41Lbecause of one or more deletions, insertions or substitutions, andexhibits the desired LAR-binding property. A41L polypeptides, variantsand derivative provided herein include, but are not limited to,fragments, analogs and variants of the native A41L proteins of SEQ IDNOs:2 through 9. Such analogs are discussed in more detail below.

The variant amino acid sequence preferably is at least 80% identical,more preferably at least 90%, most preferably at least 95% identical toa native A41L amino acid sequence (such as the sequence of SEQ ID NO:2,4, or 6. Variant nucleic acids provided herein are preferably at least80% identical, more preferably at least 90%, most preferably at least95% identical to a native A41L DNA sequence (such as the sequence of SEQID NO:1). In one embodiment of the present invention, A41L DNA and aminoacid sequences are at least 80% identical (preferably at least 90%identical, more preferably at least 95%) to the DNA sequence of SEQ IDNO:1 or the amino acid sequence presented in SEQ ID NO:2. In otherembodiments of the invention, the amino acid sequence of the A41Lprotein is at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identical to the amino acid sequence presented in SEQ ID NO:2.

Percent identity is defined as the number of aligned symbols, i.e.nucleotides or amino acids, which are identical, divided by the totalnumber of symbols in the shorter of the two sequences. The degree ofhomology (percent identity) between two sequences may be determined byusing the alignment method of Needleman and Wunsch (J. Mol. Biol.48:443, 1970) as revised by Smith and Waterman (Adv. Appl. Math 2:482,1981), with a unary comparison matrix (containing a value of 1 foridentities and 0 for nonidentities) for nucleotides, and the weightedcomparison matrix of Gribskov and Burgess (Nucl. Acids Res. 14:6745,1986) as described by Schwartz and Dayhoff (Atlas of Protein Sequenceand Structure, National Biomedical Research Foundation, pp. 353-358,1979) for amino acids.

Preferably, the comparison is done using a computer program. Anexemplary, preferred computer program is the Genetics Computer Group(GCG; Madison, Wis.) Wisconsin package version 10.0 program, ‘GAP.’ Thepreferred default parameters for the ‘GAP’ program includes: (1) The GCGimplementation of the previously stated comparison matrixes fornucleotides and amino acids; (2) a penalty of 30 for each gap and anadditional penalty of 1 for each symbol in each gap for amino acidsequences, or penalty of 50 for each gap and an additional penalty of 3for each symbol in each gap for nucleotide sequences; (3) no penalty forend gaps; and (4) no maximum penalty for long gaps. Other programs usedby one skilled in the art of sequence comparison may also be used.

Alterations of the native amino acid sequence may be accomplished by anyof a number of known techniques. Mutations can be introduced atparticular loci by synthesizing oligonucleotides containing a mutantsequence, flanked by restriction sites enabling ligation to fragments ofthe native sequence. Following ligation, the resulting reconstructedsequence encodes an analog having the desired amino acid insertion,substitution, or deletion.

Alternatively, oligonucleotide-directed site-specific mutagenesisprocedures can be employed to provide an altered gene having particularcodons altered according to the substitution, deletion, or insertionrequired. Methods of making the alterations set forth above aredisclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al.(Genetic Engineering: Principles and Methods, Plenum Press, 1981);Kunkel (Proc. Natl Acad. Sci. USA 82:488, 1985); Kunkel et al. (Methodsin Enzymol. 154:367, 1987); and U.S. Pat. Nos. 4,518,584 and 4,737,462.

Variants may comprise conservatively substituted sequences, meaning thata given amino acid residue is replaced by a residue having similarphysiochemical characteristics. Examples of conservative substitutionsinclude substitution of one aliphatic residue for another, such as Ile,Val, Leu, or Ala for one another, or substitutions of one polar residuefor another, such as between Lys and Arg; Glu and Asp; or Gln and Asn.Other such conservative substitutions, for example, substitutions ofentire regions having similar hydrophobicity characteristics, are wellknown. Conservatively substituted A41L polypeptides encompassed by thepresent invention are those that retain the ability to bind LAR. Certainembodiments of A41L proteins contain from one to ten conservative aminoacid substitutions.

One embodiment of the present invention is directed to an A4Lpolypeptidecomprising conservative substitution(s) in the amino acid sequencepresented in SEQ ID NO:2, wherein the conservatively substitutedpolypeptide exhibits a biological activity that is essentiallyequivalent to that of the native protein of SEQ ID NO:2. Those of skillin the art will be guided in selecting amino acid residues forsubstitution by comparing the amino acid to be substituted with thoseshown for various A41L proteins in FIG. 1. Thus, for example, a Cys atamino acid residue 11 of a vaccinia or variola virus A41L protein couldbe substituted with another amino acid, for example a Gly as is found inCPV. Alternatively (or additionally), the Tyr at amino acid residue 114of a variola virus A41L polypeptide could be substituted with anotheramino acid, for example, a His as is found in the corresponding locationin CPV and vaccinia A4Lpolypeptides.

A41L also may be modified to create derivatives by forming covalent oraggregative conjugates with other chemical moieties, such as glycosylgroups, lipids, phosphate, acetyl groups and the like. Covalentderivatives of A41L may be prepared by linking the chemical moieties tofunctional groups on A41L amino acid side chains or at the N-terminus orC-terminus of an A4Lpolypeptide. Other derivatives of A41L within thescope of this invention include covalent or aggregative conjugates ofA41L or its fragments with other proteins or polypeptides, such as bysynthesis in recombinant culture as N-terminal or C-terminal fusions.

An A41Lprotein may be treated or derivatized to reduce theimmunogenicity and antigenicity thereof. Such modification may bedesirable if A41L is to be administered repeatedly to an individual,e.g., to treat a chronic condition. One approach involves attaching thepolymer polyethylene glycol (PEG) to an A4Lprotein. Chemicalmodification with PEG has reduced the immunogenicity or antigenicity ofa number of proteins (See Katre, N., J. Immunol. 144:209, 1990; andDelgado et al., Critical Reviews in Therapeutic Drug Carrier Systems,9:249, 1992.) Modification with PEG has also been reported to increasethe serum half-life and solubility of certain proteins. PEG may becovalently linked to lysine residues, to carbohydrate moieties onglycosylated proteins, or selectively to the N-terminus of proteins, forexample. Modified A41L proteins can be tested in a suitable bindingassay to confirm that the desired LAR-binding property is retained.

Fragments of A41L may be less immunogenic than the corresponding fulllength proteins. The glycosylation pattern may affect the immunogenicityof a protein. As discussed above, glycosylation of recombinant proteinsmay be altered through the choice of host cells.

A41L polypeptide fusions can comprise peptides added to facilitatepurification and identification of A41L(referred to as ‘tag’ peptides).Such peptides include, for example, poly-His or the antigenicidentification peptides described in U.S. Pat. No. 5,011,912 and in Hoppet al., Bio/Technology 6:1204, 1988. Additional, useful tag proteinsinclude green fluorescent protein (GFP; Chalfie et al., Science 263:802,1994), an N-terminal peptide that contains recognition sites for amonoclonal antibody, a specific endopeptidase, and a site-specificprotein kinase (PKA; Blanar and Rutter, Science 256:1014, 1992), birA(Altman et al., Science 274:94, 1996) and glutathione S transferase(GST: Smith and Johnson, Gene 67:31, 1988).

A very useful tag peptide is the FLAG® peptide (SEQ ID NO:11), disclosedin U.S. Pat. No. 5,011,912 and in Hopp et al., which is highly antigenicand provides an epitope reversibly bound by a specific monoclonalantibody enabling rapid assay and facile purification of expressedrecombinant protein. This sequence is also specifically cleaved bybovine mucosal enterokinase at the residue immediately following anAsp-Lys pairing. Fusion proteins capped with this peptide may also beresistant to intracellular degradation in E. coli. A murine hybridomadesignated 4E11 produces a monoclonal antibody that binds the FLAG®peptide in the presence of certain divalent metal cations (as describedin U.S. Pat. No. 5,011,912) and has been deposited with the AmericanType Culture Collection under accession no HB 9259.

The present invention further includes A41L polypeptides with or withoutassociated native-pattern glycosylation. A41L expressed in yeast ormammalian expression systems (e.g., COS-7 cells) may be similar to orsignificantly different from a native A41L polypeptide in molecularweight and glycosylation pattern, depending upon the choice ofexpression system. Expression of A41L polypeptides in bacterialexpression systems, such as E. coli, provides non-glycosylatedmolecules.

DNA constructs that encode various additions or substitutions of aminoacid residues or sequences, or deletions of terminal or internalresidues or sequences not needed for biological activity or binding canbe prepared. For example, N-glycosylation sites in A41L can be modifiedto preclude glycosylation, allowing expression of a more homogeneous,reduced carbohydrate analog in mammalian and yeast expression systems.N-glycosylation sites in eukaryotic polypeptides are characterized by anamino acid triplet Asn-X-Y, wherein X is any amino acid except Pro and Yis Ser or Thr. CPV A41L comprises a potential glycosylation site atamino acid residue 71 (which corresponds to amino acid residue 70 invaccinia or variola virus). Appropriate modifications to the nucleotidesequence encoding this triplet will result in substitutions, additionsor deletions that prevent attachment of carbohydrate residues at the Asnside chain. Alteration of a single nucleotide, chosen so that Asn isreplaced by a different amino acid, for example, is sufficient toinactivate an N-glycosylation site. Known procedures for inactivatingN-glycosylation sites in proteins include those described in U.S. Pat.No. 5,071,972 and EP 276,846, hereby incorporated by reference.

In another example, codons for Cys residues that are not essential forbiological activity can be altered to cause the Cys residues to bedeleted or replaced with other amino acids. Formation of incorrectintramolecular disulfide bridges upon renaturation is thus prevented.The A41L protein of SEQ ID NOs:3 through 9 comprises nine Cys residues,and the A41L protein of SEQ ID NO:2 comprises 8 Cys residues.Accordingly, the first Cys residue (at amino acid 11 of SEQ ID 3 through9) may be deleted or substituted with another amino acid.

The present invention provides both non-naturally occurring andnaturally occurring biologically active A41L variants. Examples ofnaturally occurring variants are proteins that result from proteolyticcleavage of the A41L protein, wherein the ability to bind LAR isretained. Variations attributable to proteolysis include, for example,differences in the N- or C-termini upon expression in different types ofhost cells, due to proteolytic removal of one or more terminal aminoacids from the A41L protein (generally from 1-5 terminal amino acids).Naturally occurring variations in the DNA sequence may include silentmutations, for example, or deletions that do not result in a shift inthe reading frame.

Due to the known degeneracy of the genetic code wherein more than onecodon can encode the same amino acid, a DNA sequence may vary from thatpresented in SEQ ID NO:1, and still encode an A4Lprotein having theamino acid sequence of SEQ ID NO:2. Such variant nucleic acids mayresult from silent mutations (e.g., occurring during PCR amplification),and may be the product of deliberate mutagenesis of a native sequence.

Particular embodiments of A41L-encoding DNAs are isolated DNAscomprising nucleotides 1 to 654 or 58 to 654 of SEQ ID NO: 1. The DNAthat comprises nucleotides 1 to 654 of SEQ ID NO:1 encodes a cowpox A41Lprotein that includes the native signal peptide, whereas a DNA havingthe sequence of nucleotides 58 to 654 encodes the predicted, mature formof the protein. The invention further provides various forms of cowpoxA41L DNAs, including a DNA comprising nucleotides x to y of SEQ ID NO:1,wherein x represents an integer from 1 to 73 (preferably, from 1 to 58),inclusive, and y represents an integer from 621 to 654, inclusive.

Disclosed herein are isolated nucleic acids encoding biologically activeA41L, selected from: (a) the nucleotide sequence presented in SEQ IDNOS:1 or 3; (b) DNA capable of hybridization to a nucleotide sequence of(a) under moderately or severely stringent conditions and which encodesa biologically active A41L; and (c) DNA which is degenerate as a resultof the genetic code to the nucleotide sequence defined in (a) or (b).The A41L proteins encoded by such nucleic acids are encompassed by thepresent invention.

Nucleic acid sequences disclosed herein include isolated DNA and RNAsequences that hybridize to native A41L nucleotide sequences underconditions of moderate or severe stringency, and which encodebiologically active A41L. For use in the therapeutic methods of thepresent invention, the desired biological activity of the encoded A41Lis the ability to bind LAR. Moderately stringent hybridizationconditions refer to conditions described in, for example, Sambrook etal. Molecular Cloning: A Laboratory Manual, 2 ed. Vol. 1, pp. 1.101-104,Cold Spring Harbor Laboratory Press, (1989). Conditions of moderatestringency, as defined by Sambrook et al., include use of a prewashingsolution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0) and hybridization atabout 55 degrees C., 5×SSC, overnight, followed by washing at 50-55degrees C. in 2×SSC, 0.1% SDS. Conditions of severe stringency includehigher temperatures of hybridization and washing, in lower salt. Theskilled artisan will recognize that the temperature and wash solutionsalt concentration may be adjusted as necessary according to factorssuch as the length of the probe. Examples of A41L-encoding nucleic acidsinclude those that will hybridize to the nucleotide sequence of SEQ IDNOS:1, 3, or 5, under severely stringent conditions that includehybridization at 68 degrees C. followed by washing in 0.1×SSC/0.1% SDSat 63-68 degrees C.

Examples of A41L proteins encoded by DNA that varies from the native DNAsequence of SEQ ID NOS:1, 3, or 5, wherein the variant DNA willhybridize to the native DNA sequence under moderately or severelystringent conditions, include, but are not limited to, A41L fragmentsand A41L proteins comprising inactivated N-glycosylation site(s), orconservative amino acid substitution(s), as described above. A41Lproteins encoded by DNA derived from organisms other than cowpox virus,wherein the DNA will hybridize to the DNA of SEQ ID NO:1, are alsoencompassed.

Variants possessing the requisite ability to bind LAR may be identifiedby any suitable assay. A biosensor unit may be employed, as described inexample 2. Alternatively, biological activity of an A4Lvariant may bedetermined by competition with a native A41L (e.g., the A41L of SEQ IDNO:2) for binding to a given LAR (i.e. competitive binding assays). Onetype of a competitive binding assay employs an A41L/Fc fusion proteinbound to a solid phase through the interaction of the Fc moiety withProtein A or Protein G affixed to the solid phase. The ability of anA4Lvariant to inhibit binding of a labeled LAR to the immobilizedA41L/Fc is analyzed by conventional techniques. Variants capable ofbinding LAR find use, e.g., for LAR or treating a disease mediated byLAR.

A41L polypeptides may be employed as reagents in vitro assays. Oneexample involves the use of A41L in screening assays to detect orisolate LAR in a biological sample or cell culture. Similarly, LARpolypeptides, particularly soluble forms of LAR, may be used as reagentsin vitro assays.

Soluble forms of LAR include a peptide comprising amino acids x to y,wherein x represents an integer from 1 to 20, inclusive, and yrepresents an integer from 960 to 965, inclusive, of SEQ ID NO:10;additional soluble forms of LAR comprise amino acids x to y, wherein xrepresents an integer from 1 to 20, inclusive, and y represents aninteger from 302 to 312, inclusive, of SEQ ID NO:10, or amino acids x toy, wherein x represents an integer from 1 to 24, inclusive, and yrepresents an integer from 302 to 322, inclusive, of SEQ ID NO:10.Additional soluble forms of LAR include polypeptides comprising aminoacids x to y, wherein x represents an integer from 1 to 24, inclusive,and y represents an integer from 296 to 306, inclusive, of SEQ ID NO:12;and polypeptides comprising amino acids x to y, wherein x represents aninteger from 1 to 20, inclusive, and y represents an integer from 296 to306, inclusive, of SEQ ID NO:12. Fragments of the aforementionedpolypeptides comprising the immunoglobulin domains of LAR (amino acids44 through 294 of SEQ ID NO:10; amino acids 44 through 288 of SEQ IDNO:12) can also be prepared and evaluated for the ability to bind anA41L polypeptide.

Fragments of such polypeptides will also be useful in assays, and asantagonists of LAR in vivo or in vitro. Moreover, variants of LAR arealso comprehended herein. Such variants include those described abovefor A41L polypeptides (i.e., non-naturally occurring and naturallyoccurring biologically active LAR variants, LAR polypeptides encoded byisolated DNA and RNA sequences that hybridize to native LAR nucleotidesequences under conditions of moderate or severe stringency, and LARpolypeptides comprising various additions or substitutions of amino acidresidues or sequences, or deletions of terminal or internal residues orsequences not needed for biological activity, for example, alteration ofglycosylation sites or Cys residues not needed for biological activity).

The variant amino acid sequence preferably is at least 80% identical,more preferably at least 90%, most preferably at least 95% identical toa native LAR amino acid sequence (such as the sequence of SEQ ID NO:10or 12. Variant nucleic acids provided herein are preferably at least 80%identical, more preferably at least 90%, most preferably at least 95%identical to a native LAR DNA sequence (such as the sequence of SEQ IDNO:10 or 12). In one embodiment of the present invention, LAR DNA andamino acid sequences are at least 80% identical (preferably at least 90%identical, more preferably at least 95%) to the DNA sequence of SEQ IDNO:10 or the amino acid sequence presented in SEQ ID NO:12. In otherembodiments of the invention, the amino acid sequence of the LARpolypeptide is at least 90%, at least 91%, at least 92%, at least 93%,at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99% identical to the amino acid sequence presented in SEQ IDNOs:10 or 12. Nucleic acids encoding the aforementioned polypeptides arealso included within the scope of the invention.

Expression Systems

The present invention provides recombinant expression vectors forexpression of A41L and/or LAR polypeptides, and host cells transformedwith the expression vectors. Any suitable expression system may beemployed. The vectors include an A41L DNA or a LAR DNA sequence operablylinked to suitable transcriptional or translational regulatorynucleotide sequences, such as those derived from a mammalian, microbial,viral, or insect gene. Examples of regulatory sequences includetranscriptional promoters, operators, or enhancers, an mRNA ribosomalbinding site, and appropriate sequences which control transcription andtranslation initiation and termination. Nucleotide sequences areoperably linked when the regulatory sequence functionally relates to theA41L or LAR DNA sequence. Thus, a promoter nucleotide sequence isoperably linked to an A41L or LAR DNA sequence if the promoternucleotide sequence controls the transcription of the A41L or LAR DNAsequence. The ability to replicate in the desired host cells, usuallyconferred by an origin of replication, and a selection gene by whichtransformants are identified, may additionally be incorporated into theexpression vector.

If desired, a native signal sequence may be replaced with a heterologoussignal sequence. A signal peptide that promotes higher levels ofsecretion from a particular type of host cells than does the nativesignal peptide may be chosen, for example. A DNA sequence encoding theheterologous signal peptide (secretory leader) is fused in frame to theA41L or LAR sequence so that the encoded polypeptide is initiallytranslated as a fusion protein comprising the signal peptide. The signalpeptide is cleaved from the polypeptide upon secretion of thepolypeptide from the cell.

Suitable host cells for expression of A41L and/or LAR polypeptidesinclude prokaryotes, yeast or higher eukaryotic cells. Appropriatecloning and expression vectors for use with bacterial, fungal, yeast,and mammalian cellular hosts are described, for example, in Pouwels etal. Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., (1985).Cell-free translation systems could also be employed to produce suchpolypeptides using RNAs derived from DNA constructs disclosed herein.

Prokaryotes include gram negative or gram positive organisms, forexample, E. coli or Bacilli. Suitable prokaryotic host cells fortransformation include, for example, E. coli, Bacillus subtilis,Salmonella typhimurium, and various other species within the generaPseudomonas, Streptomyces, and Staphylococcus. In a prokaryotic hostcell, such as E. coli, an A41L or a LAR polypeptide may include anN-terminal methionine residue to facilitate expression of therecombinant polypeptide in the prokaryotic host cell. The N-terminal Metmay be cleaved from the expressed recombinant polypeptide.

Expression vectors for use in prokaryotic host cells generally compriseone or more phenotypic selectable marker genes. A phenotypic selectablemarker gene is, for example, a gene encoding a protein that confersantibiotic resistance or that supplies an autotrophic requirement.Examples of useful expression vectors for prokaryotic host cells includethose derived from commercially available plasmids such as the cloningvector pBR322 (ATCC 37017). pBR322 contains genes for ampicillin andtetracycline resistance and thus provides simple means for identifyingtransformed cells. An appropriate promoter and an A41L DNA (or LAR DNA)sequence are inserted into the pBR322 vector. Other commerciallyavailable vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and pGEM1 (Promega Biotec, Madison, Wis.,USA).

Promoter sequences commonly used for recombinant prokaryotic host cellexpression vectors include β-lactamase (penicillinase), lactose promotersystem (Chang et al., Nature 275:615, 1978; and Goeddel et al., Nature281:544, 1979), tryptophan (trp) promoter system (Goeddel et al., Nucl.Acids Res. 8:4057, 1980; and EP-A-36776) and tac promoter (Maniatis,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,p. 412, 1982). A particularly useful prokaryotic host cell expressionsystem employs a phage λ P_(L) promoter and a cI857ts thermolabilerepressor sequence. Plasmid vectors available from the American TypeCulture Collection which incorporate derivatives of the λ P_(L) promoterinclude plasmid pHUB2 (resident in E. coli strain JMB9 (ATCC 37092)) andpPLc28 (resident in E. coli RR1 (ATCC 53082)).

Recombinant polypeptides alternatively may be expressed in yeast hostcells, preferably from the Saccharomyces genus (e.g., S. cerevisiae).Other genera of yeast, such as Pichia or Kluyveromyces, may also beemployed. Yeast vectors will often contain an origin of replicationsequence from a 2μ yeast plasmid, an autonomously replicating sequence(ARS), a promoter region, sequences for polyadenylation, sequences fortranscription termination, and a selectable marker gene. Suitablepromoter sequences for yeast vectors include, among others, promotersfor metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J.Biol. Chem. 255:2073, 1980) or other glycolytic enzymes (Hess et al., J.Adv. Enzyme Reg. 7:149, 1968; and Holland et al., Biochem. 17:4900,1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase. Other suitable vectors and promoters for use in yeastexpression are further described in Hitzeman, EPA-73,657. Anotheralternative is the glucose-repressible ADH2 promoter described byRussell et al. (J. Biol. Chem. 258:2674, 1982) and Beier et al. (Nature300:724, 1982). Shuttle vectors replicable in both yeast and E. coli maybe constructed by inserting DNA sequences from pBR322 for selection andreplication in E. coli (Amp^(r) gene and origin of replication) into theabove-described yeast vectors.

The yeast α-factor leader sequence may be employed to direct secretionof the polypeptide. See, e.g., Kurjan et al., Cell 30:933, 1982; Bitteret al., Proc. Natl. Acad. Sci. USA 81:5330, 1984; U.S. Pat. No.4,546,082; and EP 324,274. Other leader sequences suitable forfacilitating secretion of recombinant polypeptides from yeast hosts areknown to those skilled in the pertinent field.

Yeast transformation protocols are known to those of skill in the art.One such protocol is described by Hinnen et al., Proc. Natl. Acad. Sci.USA 75:1929, 1978. The Hinnen et al. protocol selects for Trp⁺transformants in a selective medium, wherein the selective mediumconsists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose,10 μg/ml adenine and 20 μg/ml uracil.

Yeast host cells transformed by vectors containing ADH2 promotersequence may be grown for inducing expression in a “rich” medium. Anexample of a rich medium is one consisting of 1% yeast extract, 2%peptone, and 1% glucose supplemented with 80 μg/ml adenine and 80 μg/mluracil. Derepression of the ADH2 promoter occurs when glucose isexhausted from the medium.

Mammalian or insect host cell culture systems could also be employed toexpress recombinant polypeptides. Baculovirus systems for production ofheterologous proteins in insect cells are reviewed by Luckow andSummers, Bio/Technology 6:47, 1988. Established cell lines of mammalianorigin also may be employed. Examples of suitable mammalian host celllines include the COS-7 line of monkey kidney cells (ATCC CRL 1651)(Gluzman et al., Cell 23:175, 1981), L cells, C127 cells, 3T3 cells(ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, and BHK(ATCC CRL 10) cell lines, and the CV-1/EBNA-1 cell line derived from theAfrican green monkey kidney cell line CVI (ATCC CCL 70) as described byMcMahan et al. (EMBO J. 10: 2821, 1991).

Transcriptional and translational control sequences for mammalian hostcell expression vectors may be excised from viral genomes. Commonly usedpromoter sequences and enhancer sequences are derived from Polyomavirus, Adenovirus 2, Simian Virus 40 (SV40), and human cytomegalovirus.DNA sequences derived from the SV40 viral genome, for example, SV40origin, early and late promoter, enhancer, splice, and polyadenylationsites may be used to provide other genetic elements for expression of astructural gene sequence in a mammalian host cell. Viral early and latepromoters are particularly useful because both are easily obtained froma viral genome as a fragment which may also contain a viral origin ofreplication (Fiers et al., Nature 273:113, 1978). Smaller or larger SV40fragments may also be used, provided the approximately 250 bp sequenceextending from the Hind III site toward the Bgl I site located in theSV40 viral origin of replication site is included.

Expression vectors for use in mammalian host cells can be constructed asdisclosed by Okayama and Berg (Mol. Cell. Biol. 3:280, 1983). A usefulsystem for stable high level expression of mammalian cDNAs in C127murine mammary epithelial cells is described by Cosman et al. (Mol.Immunol. 23:935, 1986). A high expression vector, PMLSV N1/N4, describedby Cosman et al., Nature 312:768, 1984 has been deposited as ATCC 39890.Additional suitable mammalian expression vectors are described inEP-A-0367566, and in PCT application WO 91/18982. In one embodiment, thevectors are derived from retroviruses.

A native A41L signal peptide is employed in the expression systemdescribed in example 1. Alternatively, DNA encoding a heterologoussignal sequence (e.g., derived from a mammalian protein) may be added.Examples include the signal sequence for interleukin-7 (IL-7) describedin U.S. Pat. No. 4,965,195; the signal sequence for interleukin-2receptor described in Cosman et al., Nature 312:768, 1984; theinterleukin-4 receptor signal peptide described in EP 367,566; the typeI interleukin-1 receptor signal peptide described in U.S. Pat. No.4,968,607; and the type II interleukin-1 receptor signal peptidedescribed in EP 460,846.

A41L and LAR Proteins and Uses Thereof

The present invention provides purified A41L polypeptides, which may beproduced by recombinant expression systems as described above orpurified from naturally occurring, virally infected cells. Recombinantexpression systems may be preferred for purification of certain A41Lproteins due to the infective nature of the virus from which they arederived. The invention further provides purified LAR polypeptides, whichmay be produced by recombinant expression systems as described above.The desired degree of purity depends on the intended use of the protein.A relatively high degree of purity is desired when the protein is to beadministered in vivo, for example.

Advantageously, the polypeptides of the invention are purified such thatno protein bands corresponding to other proteins are detectable bySDS-polyacrylamide gel electrophoresis (SDS-PAGE). It will be recognizedby one skilled in the pertinent field that multiple bands correspondingto A41L protein (or LAR protein) may be detected by SDS-PAGE, due todifferential glycosylation, variations in post-translational processing,and the like. A preparation of A41L protein is considered to be purifiedas long as no bands corresponding to different (non-A41L) proteins arevisualized; similarly, for preparations of LAR proteins, as no bandscorresponding to different (non-LAR) proteins are visualized. Theproteins of the invention are most preferably purified to substantialhomogeneity, as indicated by a single protein band upon analysis bySDS-PAGE. The protein band may be visualized by silver staining,Coomassie blue staining, or (if the protein is radiolabeled) byautoradiography.

One process for producing the polypeptides of the invention comprisesculturing a host cell transformed with an expression vector comprising aDNA sequence that encodes said polypeptides under conditions such that adesired polypeptide (A41L or LAR) is expressed. The protein is thenrecovered from the cell culture, using standard procedures.Advantageously, the expression vector encodes a signal peptide fused tothe N-terminus of the protein, such that the protein is secreted fromthe host cell and may be recovered from the culture medium.

For example, the culture medium first may be concentrated using acommercially available protein concentration filter, for example, anAmicon or Millipore Pellicon ultrafiltration unit. Following theconcentration step, the concentrate can be applied to a purificationmatrix such as a gel filtration medium. Alternatively, an anion exchangeresin can be employed, for example, a matrix or substrate having pendantdiethylaminoethyl (DEAE) groups. The matrices can be acrylamide,agarose, dextran, cellulose or other types commonly employed in proteinpurification. Alternatively, a cation exchange step can be employed.Suitable cation exchangers include various insoluble matrices comprisingsulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred.Finally, one or more reversed-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,(e.g., silica gel having pendant methyl or other aliphatic groups) canbe employed to further purify A41L. Some or all of the foregoingpurification steps, in various combinations, can be employed to providea substantially homogeneous recombinant protein.

It is also possible to utilize an immunoaffinity column containing anantibody that binds A41L to purify this polypeptide. Example 5 describesa procedure for employing an A41L protein to generate monoclonalantibodies reactive therewith. Similar procedures could be used togenerate monoclonal antibodies that bind LAR, which could then be usedin affinity purification thereof. Moreover, A41L can be used toaffinity-purify LAR, and LAR can be used to affinity purify A41L, basedon their interaction.

Recombinant protein produced in bacterial culture is usually isolated byinitial disruption of the host cells, centrifugation, extraction fromcell pellets if an insoluble polypeptide, or from the supernatant fluidif a soluble polypeptide, followed by one or more concentration,salting-out, ion exchange, affinity purification or size exclusionchromatography steps. Finally, RP-HPLC can be employed for finalpurification steps. Microbial cells can be disrupted by any convenientmethod, including freeze-thaw cycling, sonication, mechanicaldisruption, or use of cell lysing agents.

Transformed host cells are preferably employed to express A41L (or LAR)as a secreted polypeptide. This simplifies purification. Secretedrecombinant polypeptide from a host cell can be purified by methodsanalogous to those disclosed by Urdal et al. (J. Chromatog. 296:171,1984). Urdal et al. describe two sequential, reversed-phase HPLC stepsfor purification of recombinant human IL-2 on a preparative HPLC column.

In one embodiment of the invention, non-recombinant A41L is purified byprocedures analogous to those described herein for recombinant A41Lproteins. Briefly, suitable cells (e.g., rabbit kidney cells or CV1cells) are infected with cowpox virus, vaccinia virus (for example,strain Copenhagen, or another strain) or variola virus (for example,strain India-1966 or another strain), and incubated to allow secretionof A41L from the cells. The culture supernatant is collected, cells areremoved (e.g., by centriftigation), and the supernatant is applied to asuitable purification resin (for example, an antibody affinity column).Proteins are eluted from the column by suitable methods, and thefraction containing the desired A41L protein is recovered.

Certain uses of A41L polypeptides flow from the ability to bind LAR. Onesuch use of A41L is as a reagent in protein purification procedures.A41L or A41L/Fc fusion proteins may be attached to a solid supportmaterial by conventional techniques and used to purify LAR by affinitychromatography. This use is illustrated in example 2.

A41L also finds use in modulating the biological activity of LAR. Thisuse stems from the unexpected finding that A41L binds to LAR, asreported in example 3.

The present invention provides a method of treating a disorder mediatedby LAR, comprising administering an effective amount of an A41Lpolypeptide to a mammal afflicted with such a condition. Any of theforms of A41L polypeptides described herein may be employed, including,but not limited to, native A41L proteins and variants, derivatives,oligomers, and biologically active fragments thereof, as well as fusionproteins comprising A41L. In particular embodiments of the invention,the mature form of the cowpox A41L of SEQ ID NO:2, the vaccinia virusA41L of SEQ ID NOs:4 through 7, or the variola (smallpox) A41L of SEQ IDNOs:8 through 10 is employed.

A disorder is said to be mediated by LAR if LAR causes or promotes, atleast in part, or exacerbates the disorder. LAR may cause a conditionindirectly, for example by modulating one or more pathways that regulatecytokine receptor signaling. Alternatively, LAR may cause or promote acondition in a more direct manner, for example via its interaction withthe insulin receptor.

Because A41L is a virulence factor (that is, it is required forproliferation of a poxvirus in a host animal but not for proliferationof a poxvirus in vitro), it plays a role in regulating the immune and/orinflammatory response. A role for LAR in modulating a host immune orinflammatory response is supported by the finding that itdephosphorylates JAK2, similar to CD45 (Irie-Sasaki et al., supra).Accordingly, A41L will have use in modulating an immune and/orinflammatory response. Moreover, the interaction of A41L and LAR servesas mechanism by which to identify additional regulators of an immuneand/or inflammatory response, as described herein. Compounds thatinhibit the interaction of A41L and LAR (identified as described herein)will also be useful in treating or ameliorating poxvirus infections bypreventing the virulence factor (A41L) from binding its counterstructurein the infected host.

Moreover, the expression and/or activity of PTPs, including LAR, isknown to be increased in obesity and Type 2 diabetes (see, for example,Zabolotny et al., Proc. Natl. Acad. Sci. USA 98:5187, 2001).Accordingly, the interaction of A41L and LAR will be useful inidentifying compounds that can be used to regulate insulin signaling,which will be useful in treatment of conditions in which theover-expression or hyperactivity of PTPs plays a role (i.e., obesityand/or insulin resistance).

Additionally, a potential role for LAR in the proliferation of cells mayalso indicate a use for a method that employs the interaction of LAR andA41L to identify compounds that regulate cell proliferation. Forexample, the association of the region of the human chromosome to whichLAR has been mapped with tumors of neuroectodermal origin and theassociation of changes in expression of LAR with the ability of cells toproliferate suggest that a compound that modulates the activity of LARmay be useful in regulating cell growth (either in tumor cells or neuralcells). Moreover, differential splicing of LAR appears to bedevelopmentally regulated. Accordingly, reagents specific for certainforms of LAR will be useful in diagnostic or therapeutic applicationsrelevant to the particular form for which they are specific.

The A41L (or other, identified compound) preferably is administered inthe form of a pharmaceutical composition. Such compositions can beformulated according to known methods that are used to preparepharmaceutically useful compositions. Components that are commonlyemployed in pharmaceutical formulations are described in Remington'sPharmaceutical Sciences, 16th ed., 1980, Mack Publishing Company, forexample.

Compositions of the present invention comprise an effective amount of apurified compound (i.e., a purified A41L polypeptide, a purified LARpolypeptide, or a compound that modulates the interaction of A41L andLAR) and a suitable (e.g., pharmaceutically acceptable) diluent,excipient, or carrier. Such carriers will be essentially nontoxic(minimally toxic) to patients at the dosages and concentrationsemployed. The composition may additionally include a suitable emulsifieror preservative. Ordinarily, the preparation of such compositions mayentail combining an A4Lpolypeptide with buffers, antioxidants such asascorbic acid, low molecular weight (less than about 10 residues)peptides, proteins, amino acids, carbohydrates including glucose,sucrose, or dextrans, chelating agents such as EDTA, glutathione, orother stabilizers and excipients. Neutral buffered saline is oneappropriate diluent. Pharmaceutical compositions suitable for inhalationare among the compositions contemplated herein.

Useful compounds may be incorporated into polymeric compounds (such aspolyacetic acid, polyglycolic acid, hydrogels, dextran, etc.) orincorporated into liposomes, microemulsions, micelles, unilamellar ormultilamellar vesicles, erythrocyte ghosts or spheroblasts. Suchcompositions will influence the physical state, solubility, stability,rate of in vivo release, and rate of in vivo clearance of A41 L, and arethus chosen according to the intended application.

For therapeutic use, the compositions are administered in a manner anddosage appropriate to the indication and the patient. The amountadministered will be effective in ameliorating the condition, bymodulating (inhibiting or increasing, as needed for the indication) theactivity of LAR in vivo. As will be understood by one skilled in thepertinent field, a therapeutically effective dosage will vary accordingto such factors as the nature and severity of the condition, thelocation of affected tissue (e.g., the site of inflammation) within thebody, and the age, condition and size of the patient. Administration maybe by any suitable route, depending on the nature of the disorder,including but not limited to intravenous or local injection, inhalation,continuous infusion, local infusion during surgery, or sustained releasefrom implants (such as gels, membranes, and the like).

Oligomeric Forms of A41L and/or LAR Polypeptides

The present invention encompasses polypeptides (either A41L or LAR) inthe form of oligomers, such as dimers, trimers, or higher oligomers.Oligomers may be formed by disulfide bonds between cysteine residues ondifferent polypeptides, for example. In other embodiments, oligomerscomprise from two to four polypeptides joined by covalent ornon-covalent interactions between peptide moieties fused to thepolypeptides. Such peptide moieties may be peptide linkers (spacers), orpeptides that have the property of promoting oligomerization. Leucinezippers and certain polypeptides derived from antibodies are among thepeptides that can promote oligomerization of polypeptides attachedthereto. Nucleic acids encoding oligomers, or fusion proteins that arecomponents of such oligomers, are provided herein.

Preparation of fusion proteins comprising heterologous polypeptidesfused to various portions of antibody-derived polypeptides (includingthe Fc domain) has been described, e.g., by Ashkenazi et al. (PNAS USA88:10535, 1991), Byrn et al. (Nature 344:667, 1990), and Hollenbaugh andAruffo (“Construction of Immunoglobulin Fusion Proteins”, in CurrentProtocols in Immunology, Suppl. 4, pages 10.19.1-10.19-11, 1992), herebyincorporated by reference. In one embodiment of the invention, an A41L(or LAR) dimer is created by fusing A41L (or LAR, respectively) to an Fcregion polypeptide derived from an antibody (yielding a fusion protein),in a manner that does not interfere with binding of A41L to LAR. A genefusion encoding the fusion protein is inserted into an appropriateexpression vector. The fusion proteins are allowed to assemble much likeantibody molecules, whereupon interchain disulfide bonds form between Fcpolypeptides, yielding divalent A41L.

One suitable Fc polypeptide is the native Fc region polypeptide derivedfrom a human IgG1, which is described in PCT application WO 93/10151,hereby incorporated by reference. Another useful Fc polypeptide is theFc mutein described in U.S. Pat. No. 5,457,035. The amino acid sequenceof the mutein is identical to that of the native Fc sequence presentedin WO 93/10151, except that amino acid 19 has been changed from Leu toAla, amino acid 20 has been changed from Leu to Glu, and amino acid 22has been changed from Gly to Ala. This mutein Fc exhibits reducedaffinity for immunoglobulin receptors.

In other embodiments, A41L (or LAR) may be substituted for the variableportion of an antibody heavy or light chain. If fusion proteins are madewith both heavy and light chains of an antibody, it is possible to forman oligomer with as many as four A41L (or LAR) polypeptides.

Alternatively, oligomeric A41L (or LAR) may comprise two or more A41L(or LAR) polypeptides joined through peptide linkers. Examples includethose peptide linkers described in U.S. Pat. No. 5,073,627 (herebyincorporated by reference). Fusion proteins comprising multiplepolypeptides separated by peptide linkers may be produced usingconventional recombinant DNA technology.

Another method for preparing oligomers involves use of a leucine zipper.Leucine zipper domains are peptides that promote oligomerization of theproteins in which they are found. Leucine zippers were originallyidentified in several DNA-binding proteins (Landschulz et al., Science240:1759, 1988), and have since been found in a variety of differentproteins. Among the known leucine zippers are naturally occurringpeptides and derivatives thereof that dimerize or trimerize. Examples ofleucine zipper domains suitable for producing A41L oligomers are thosedescribed PCT application WO 94/10308, hereby incorporated by reference.Recombinant fusion proteins comprising a polypeptide (for example, A41Lor LAR) fused to a peptide that dimerizes or trimerizes in solution areexpressed in suitable host cells, and the resulting soluble oligomericA41L is recovered from the culture supernatant.

Antibodies

Antibodies that are immunoreactive with the polypeptides disclosedherein are also provided, including antibodies that bind A41Lpolypeptide, and antibodies that bind LAR polypeptides. Such antibodiesspecifically bind to the respective polypeptides via the antigen-bindingsites of the antibody (as opposed to non-specific binding). Thus, thepolypeptides, fragments, variants, fusion proteins, etc., as set forthabove may be employed as “immunogens” in producing antibodiesimmunoreactive therewith. More specifically, the polypeptides, fragment,variants, fusion proteins, etc. contain antigenic determinants orepitopes that elicit the formation of antibodies.

These antigenic determinants or epitopes can be either linear orconformational (discontinuous). Linear epitopes are composed of a singlesection of amino acids of the polypeptide, while conformational ordiscontinuous epitopes are composed of amino acids sections fromdifferent regions of the polypeptide chain that are brought into closeproximity upon protein folding (C. A. Janeway, Jr. and P. Travers,Immuno Biology 3:9 (Garland Publishing Inc., 2nd ed. 1996)). Becausefolded proteins have complex surfaces, the number of epitopes availableis quite numerous; however, due to the conformation of the protein andsteric hindrances, the number of antibodies that actually bind to theepitopes is less than the number of available epitopes (C. A. Janeway,Jr. and P. Travers, Immuno Biology 2:14 (Garland Publishing Inc., 2nded. 1996)). Epitopes may be identified by any of the methods known inthe art.

Thus, one aspect of the present invention relates to the antigenicepitopes of the polypeptides of the invention. Such epitopes are usefulfor raising antibodies, in particular monoclonal antibodies, asdescribed in more detail below. Additionally, epitopes from thepolypeptides of the invention can be used as research reagents, inassays, and to purify specific binding antibodies from substances suchas polyclonal sera or supernatants from cultured hybridomas. Suchepitopes or variants thereof can be produced using techniques well knownin the art such as solid-phase synthesis, chemical or enzymatic cleavageof a polypeptide, or using recombinant DNA technology. An exemplaryepitope is the six-amino acid insert represented by amino acid 181through 186 of SEQ ID NO:10, which does not appear to be present inother, alternatively-spliced forms of LAR. Antibodies that recognizethis epitope would thus be specific for the form of LAR depicted in SEQID NO:10, and would not bind other forms.

As to the antibodies that can be elicited by the epitopes of thepolypeptides of the invention, whether the epitopes have been isolatedor remain part of the polypeptides, both polyclonal and monoclonalantibodies may be prepared by conventional techniques. See, for example,Monoclonal Antibodies, Hybridomas: A New Dimension in BiologicalAnalyses, Kennet et al. (eds.), Plenum Press, New York (1980); andAntibodies: A Laboratory Manual, Harlow and Land (eds.), Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., (1988).

Hybridoma cell lines that produce monoclonal antibodies specific for thepolypeptides of the invention are also contemplated herein. Suchhybridomas may be produced and identified by conventional techniques.One method for producing such a hybridoma cell line comprises immunizingan animal with a polypeptide or a DNA encoding a polypeptide; harvestingspleen cells from the immunized animal; fusing said spleen cells to amyeloma cell line, thereby generating hybridoma cells; and identifying ahybridoma cell line that produces a monoclonal antibody that binds thepolypeptide. The monoclonal antibodies may be recovered by conventionaltechniques.

The monoclonal antibodies of the present invention include chimericantibodies, e.g., humanized versions of murine monoclonal antibodies.Such humanized antibodies may be prepared by known techniques and offerthe advantage of reduced immunogenicity when the antibodies areadministered to humans. In one embodiment, a humanized monoclonalantibody comprises the variable region of a murine antibody (or just theantigen binding site thereof) and a constant region derived from a humanantibody. Alternatively, a humanized antibody fragment may comprise theantigen binding site of a murine monoclonal antibody and a variableregion fragment (lacking the antigen-binding site) derived from a humanantibody. Procedures for the production of chimeric and furtherengineered monoclonal antibodies include those described in Riechmann etal. (Nature 332:323, 1988), Liu et al. (Proc. Natl. Acad. Sci. USA84:3439, 1987), Larrick et al. (Bio/Technology 7:934, 1989), and Winterand Harris (TIPS 14:139, May, 1993). Procedures to generate antibodiestransgenically can be found in GB 2,272,440, U.S. Pat. Nos. 5,569,825and 5,545,806 and related patents claiming priority therefrom, all ofwhich are incorporated by reference herein.

Antigen-binding fragments of the antibodies, which may be produced byconventional techniques, are also encompassed by the present invention.Examples of such fragments include, but are not limited to, Fab andF(ab′)2 fragments. Antibody fragments and derivatives produced bygenetic engineering techniques are also provided.

In one embodiment, the antibodies are specific for the polypeptides ofthe present invention and do not cross-react with other proteins.Screening procedures by which such antibodies may be identified are wellknown, and may involve immunoaffinity chromatography, for example.

The antibodies of the invention can be used in assays to detect thepresence of the polypeptides or fragments of the invention, either invitro or in vivo. The antibodies also may be employed in purifyingpolypeptides or fragments of the invention by immunoaffinitychromatography.

In one embodiment of the invention, antibodies to LAR are antagonistic,that is, they bind LAR and prevent the binding of a counterstructure toLAR, thereby inhibiting the activity of LAR. Such antagonisticantibodies to LAR would be useful in upregulating an immune orinflammatory response (for example, in an individual suffering from orat risk for infection by a pathogenic or opportunistic organism, as avaccine adjuvant, and/or in treatment of cancer). Antagonists of LAR arealso useful decreasing other undesirable activities of LAR, for example,in conditions where insulin resistance plays a role (for example,obesity, Type 2 diabetes, etc.).

In another embodiment of the invention, antibodies to LAR are agonistic,that is, they bind LAR and stimulate the activity of LAR. Such agonisticantibodies to LAR would be useful in downregulating an immune orinflammatory response (for example, in autoimmune or inflammatorydisease, including atherosclerosis, arthritis, multiple sclerosis (MS),systemic lupus erythematosous (SLE), thrombosis, graft versus hostdisease, graft rejection and sepsis). Agonists of LAR will also findutility in increasing the activity of LAR in conditions wherein LARactivity is decreased (for example, in some tumors, or at certain pointsduring neural development).

Nucleic Acid Fragments

The present invention further provides fragments of the A41L nucleotidesequences presented herein. Such fragments desirably comprise at leastabout 17 consecutive nucleotides, more preferably at least 30consecutive nucleotides of the sequence presented in SEQ ID NO:1, orDNAs encoding the peptides of SEQ ID NOs:3 through 9. DNA and RNAcomplements of said fragments are provided herein, along with bothsingle-stranded and double-stranded forms of A4IL DNA.

Among the uses of such A41L nucleic acid fragments is use as a probe.Such probes may be employed in cross-species hybridization procedures toisolate A41L DNA from additional viral strains. The probes also find usein detecting the presence of A41L nucleic acids in in vitro assays andin such procedures as Northern and Southern blots. Cell types expressingA41L can be identified. Such procedures are well known, and the skilledartisan can choose a probe of suitable length, depending on theparticular intended application. The probe may be labeled (e.g., with³²P) by conventional techniques.

A41L nucleic acid fragments also find use as primers in a polymerasechain reaction (PCR). 5′ and 3′ primers corresponding to the termini ofa desired A41L DNA sequence are employed in isolating and amplifying theDNA, using conventional PCR techniques.

Similarly, LAR nucleic acid fragments will also find use as probes orprimers, and will preferably comprise at least about 17 consecutivenucleotides, more preferably at least 30 consecutive nucleotides of thesequence encoding LAR. For example, a probe comprising the nucleic acidof SEQ ID NO:13 can be used to detect the presence of nucleic acidsencoding the unique splice variant of SEQ ID NO:10 in specimens, while aprimer comprising SEQ ID NO:13 can be used to isolate and/or amplifysuch DNAs, as described previously.

Screening Assays Utilizing A41L and LAR

Specific screening methods are known in the art and along withintegrated robotic systems and collections of chemical compounds/naturalproducts are extensively incorporated in high throughput screening sothat large numbers of test compounds can be tested for antagonist oragonist activity within a short amount of time. These methods includehomogeneous assay formats such as fluorescence resonance energytransfer, fluorescence polarization, time-resolved fluorescenceresonance energy transfer, scintillation proximity assays, reporter geneassays, fluorescence quenched enzyme substrate, chromogenic enzymesubstrate and electrochemiluminescence, as well as more traditionalheterogeneous assay formats such as enzyme-linked immunosorbant assays(ELISA) or radioimmunoassays. Also comprehended herein are cell-basedassays, for example those utilizing reporter genes, as well asfunctional assays that analyze the effect of an antagonist or agonist onbiological function(s).

Moreover, combinations of screening assays can be used to find moleculesthat regulate the biological activity of A41L and/or LAR. Molecules thatregulate the biological activity of a polypeptide may be useful asagonists or antagonists of the peptide. In using combinations of variousassays, it is usually first determined whether a candidate moleculebinds to a polypeptide by using an assay that is amenable to highthroughput screening. Binding candidate molecules identified in thismanner are then added to a biological assay to determine biologicaleffects. Molecules that bind and that have an agonistic or antagonisticeffect on biologic activity will be useful in treating or preventingdisease or conditions with which the polypeptide(s) are implicated.

Homogeneous assays are mix-and-read style assays that are very amenableto robotic application, whereas heterogeneous assays require separationof free from bound analyte by more complex unit operations such asfiltration, centrifugation or washing. These assays are utilized todetect a wide variety of specific biomolecular interactions (includingprotein-protein, receptor-ligand, enzyme-substrate, and so on), and theinhibition thereof by small organic molecules. These assay methods andtechniques are well known in the art (see, e.g., High ThroughputScreening: The Discovery of Bioactive Substances, John P. Devlin (ed.),Marcel Dekker, New York, 1997 ISBN: 0-8247-0067-8). The screening assaysof the present invention are amenable to high throughput screening ofchemical libraries and are suitable for the identification of smallmolecule drug candidates, antibodies, peptides, and other antagonistsand/or agonists, natural or synthetic.

One such assay is based on fluorescence resonance energy transfer (FRET;for example, HTRF®, Packard Instrument Company, Meriden, Conn.; LANCE™,PerkinElmer LifeSciences, Wallac Oy., Turku, Finland) between twofluorescent labels, an energy donating long-lived chelate label and ashort-lived organic acceptor. The energy transfer occurs when the twolabels are brought in close proximity via the molecular interactionbetween A41L and LAR. In a FRET assay for detecting inhibition of thebinding of A41L and LAR, europium chelate or cryptate labeled A41L orLAR serves as an energy donor and streptavidin-labeled allophycocyanin(APC) bound to the appropriate binding partner (i.e., A41L if LAR islabeled, or LAR if A41L is labeled) serves as an energy acceptor. OnceA41L binds LAR, the donor and acceptor molecules are brought in closeproximity, and energy transfer occurs, generating a fluorescent signalat 665 nm. Antagonists of the interaction of A41L and LAR will thusinhibit the fluorescent signal, whereas agonists of this interactionwould enhance it.

Another useful assay is a bioluminescence resonance energy transfer, orBRET, assay, substantially as described in Xu et al., Proc. Nat. Acad.Sci. USA 96:151 (1999). Similar to a FRET assay, BRET is based on energytransfer from a bioluminescent donor to a fluorescent acceptor protein.However, a green fluorescent protein (GFP) is used as the acceptormolecule, eliminating the need for an excitation light source. ExemplaryBRET assays include BRET and BRET² from Packard BioScience, Meriden,Conn.

DELFIA® (dissociated enhanced lanthanide fluoroimmunoassay; PerkinElmerLifeSciences, Wallac Oy., Turku, Finland) is a solid-phase assay basedon time-resolved fluorometry analysis of lanthanide chelates (see, forexample, U.S. Pat. No. 4,565,790, issued Jan. 21, 1986). For this typeof assay, microwell plates are coated with a first protein (A4IL orLAR). The binding partner (LAR or A41L, respectively) is conjugated toeuropium chelate or cryptate, and added to the plates. After suitableincubation, the plates are washed and a solution that dissociateseuropium ions from solid phase bound protein, into solution, to formhighly fluorescent chelates with ligands present in the solution, afterwhich the plates are read using a reader such as a VICTOR^(2 TM)(PerkinElmer LifeSciences, Wallac Oy., Turku, Finland) plate reader todetect emission at 615 nm).

Another assay that will be useful in the inventive methods is aFlashPlate® (Packard Instrument Company, IL)-based assay. This assaymeasures the ability of compounds to inhibit protein-proteininteractions. FlashPlates® are coated with a first protein (either A41Lor LAR), then washed to remove excess protein. For the assay, compoundsto be tested are incubated with the second protein (A41L, if the platesare coated with LAR, or LAR if plates are coated with A41L) and ¹²⁵Ilabeled antibody against the second protein and added to the plates.After suitable incubation and washing, the amount of radioactivity boundis measured using a scintillation counter (such as a MicroBeta® counter;PerkinElmer LifeSciences, Wallac Oy., Turku, Finland).

The AlphaScreen™ assay (Packard Instrument Company, Meriden, Conn.).AlphaScreen™ technology is an “Amplified Luminescent ProximityHomogeneous Assay” method utilizing latex microbeads (250 nm diameter)containing a photosensitizer (donor beads), or chemiluminescent groupsand fluorescent acceptor molecules (acceptor beads). Upon illuminationwith laser light at 680 nm, the photosensitizer in the donor beadconverts ambient oxygen to singlet-state oxygen. The excitedsinglet-state oxygen molecules diffuse approximately 250 nm (one beaddiameter) before rapidly decaying. If the acceptor bead is in closeproximity to the donor bead (i.e., by virtue of the interaction of A41Land LAR), the singlet-state oxygen molecules reacts withchemiluminescent groups in the acceptor beads, which immediatelytransfer energy to fluorescent acceptors in the same bead. Thesefluorescent acceptors shift the emission wavelength to 520-620 nm,resulting in a detectable signal. Antagonists of the interaction of A41Land LAR will thus inhibit the shift in emission wavelength, whereasagonists of this interaction would enhance it.

One embodiment of a method for identifying molecules which inhibit orantagonize an activity mediated by LAR involves adding a candidatemolecule to a medium which contains cells that express A41L and LAR;changing the conditions of said medium so that, but for the presence ofthe candidate molecule, A41L would be bound to LAR, and observing thebinding and stimulation or inhibition of a functional response. Theactivity of the cells that were contacted with the candidate moleculemay then be compared with the identical cells that were not contactedand antagonists and agonists of the polypeptides of the instantinvention may be identified. The measurement of biological activity maybe performed by a number of well-known methods such as measuring theamount of protein present (e.g. an ELISA) or of the protein's activity.A decrease in biological stimulation or activation would indicate anantagonist. An increase would indicate an agonist.

Overall A41L Polyneptide Structure

U.S patent application Ser. No. 09/697,872, filed Oct. 26, 2000(incorporated by reference herein), discloses the cowpox virus (cpv) p35polypeptide structure, which has the shape of a compact globular proteinof approximately 55Å×35Å×30Å. The molecule is composed primarily of twoβ sheets that are parallel to each other, two short αhelices and largeloops connecting these secondary structure elements. Eight cysteines areinvolved in four disulfide bridges (Cys8-Cys185, Cys38-Cys223,Cys79-Cys124, Cys132-Cys171). The unfolding temperature of the cpv p35molecule is 86° C. and the four disulfide bridges probably contribute tothis high thermal stability. With the exception of Cys8, all thecysteines are located at the N- or C-terminus of beta strands. A serinerich sequence is part of a random coil structure at the N-terminus ofthe p35 molecule. The crystal structure of p35 (also referred to asviral CC-chemokine inhibitor or vCCI) was determined as described byCarfi et al., Proc. Natl. Acad. Sci. USA 96:12379, 1999.

The beta sandwich in which p35 polypeptides are folded is a structure ofnew topology, reminiscent of the collagen-binding domain fromStaphylococcus aureus adhesin. Based on the presence of eight conservedcysteines in A41L, and the alignment of these residues with the eightconserved cysteines found in p35, A41L will have topology and structuresimilar to that of p35. Accordingly, the sequence of A41L was submittedto GeneFold (Tripos, Inc., St. Louis, Mo.; Berman et al., Nucleic AcidsRes 28:235, 2000), a protein threading program that overlays a queryprotein sequence onto structural representatives of the Protein DataBank (PDB) (Jaroszewski et al., Prot Sci 7:1431, 1998). This produced avery strong hit (999.9 in all three score types) to p35, indicating thatA41L and P35 were structurally compatible and a structural model of A41Lcould be produced based on the structure of p35.

The Modeler software package from Molecular Simulations Inc. was used tocreate a family of ten structures. The structure model with the lowestprobability density function (PDF) was chosen to represent the structurefamily; these results indicated that A41L and p35 do share significantstructural and topological similarity.

When the amino acid sequences from nine different poxvirus A41L peptidesare aligned, as shown in FIG. 1, it can be seen that very few deletionsand insertions are required to align the A41L polypeptide sequences fromthe various sources. The poxvirus A41L polypeptides have at least about90% sequence identity to CPV A41L, and all have the same number andpattern of conserved cysteines in the mature protein.

In view of the close alignment of the various A41L polypeptide shown inFIG. 1, the superimposable models, and the structural similarity of A41Lwith p35, the molecular coordinates provided in U.S.S.N. 09/697,872,defining the detailed molecular architecture of the cowpox virus (cpv)p35 polypeptide, are directly applicable to the molecular architectureof A41L and the analogous gene product of other poxviruses, includingbut not limited to, Variola virus (including strains India-1967,Garcia-1966, and Harvey), vaccinia virus (including Copenhagen, TianTan, Ankara and WR strains), as well as rabbitpox virus, Camelpox virus,Ectromelia (mousepoxvirus), and Fowlpox virus. Moreover, the moleculararchitecture of the analogous gene products in any other members of thepoxvirus family will also be similar.

Moreover, knowledge of the molecular structure of the class of proteinsrepresented by p35 and A41L will allow identification of which residuesare important in binding different binding partners (or cognates). Justas definition of framework regions versus complementarity-determiningregions in antibody molecules has facilitated the design and productionof recombinant antibodies having desired characteristics (i.e.,humanized antibodies that bind with high affinity to selected antigens),understanding of the molecular structure of the proteins describedherein will facilitate design of molecules that mimic or inhibit theinteractions of A41Land/or p35 (or related) proteins with proteinsexpressed by host cells.

Such design can be accomplished either by utilizing the moleculesthemselves in screening large numbers of compounds, or in computationalapproaches (also known as de novo drug or rational drug design). Thecrystal structure coordinates of a polypeptide are input for a computerprogram, such as DOCK. Programs such as DOCK output a list of smallmolecule structures that are expected to associate with a bindingpartner as determined by their ability to compete with viralpolypeptides for binding with binding partners and their nativecognates. Ultimately, molecules identified by the DOCK program can besynthesized and screened using appropriate binding assays.

Useful molecules may bind only A41L; such molecules are expected to haveutility in modulating the binding of A41L and LAR. Molecules that bindboth A41L and p35 can also be identified; they may be particularlyuseful as anti-viral agents (i.e., by binding to both pox virusvirulence factors, A41L and p35).

Computer Analysis of Structure and/or Assay Results

In one aspect of the invention, the assays of the invention are used toidentify compounds that alter an activity of LAR. In another aspect ofthe invention, the assays of the invention are used to identifycompounds that alter an activity of A41L. The benefits of integratedrobotic systems used to analyze collections of chemicalcompounds/natural products in such assays, which preferably incorporatehigh-throughput screening methods, are most often realized by the use ofsophisticated computer and statistical techniques to manage theresulting data. In one form, the information generated in the inventivescreening assays is stored (or compiled) in electronic form, using acomputerized database that allows information to be efficientlycatalogued and retrieved. Such databases are comprised of records,usually one record for each compound, that includes information aboutthe compound, such as chemical name, structure, source, activity in abinding assay, activity in a biological assay, etc. Similar databasesmay be developed by applying computer modeling techniques based on thestructure of A41 L.

The information may be entered into the database manually, that is by auser entering data through a user interface (i.e., keyboard, touchpad,etc.), or it may be entered electronically as in when a robotic systemfor analysis of compounds generates electronic results that aretransferred to another computer system (often referred to as uploading).Such information is usually stored in a discrete area of the recordreferred to as a field. Additionally, the information, preferably in theform of a database, may be stored permanently or temporarily on variousforms of storage media, including paper, compact disks, floppy disks,magnetic tapes, optical tapes, hard drives, computer system memoryunits, and the like.

The database may be stand-alone, or the records therein may be relatedto other databases (a relational database). Examples of other databasesinclude publicly available, well-known databases such as GenBank forpeptides and nucleic acids (and associated databases maintained by theNational Center for Biotechnology Information or NCBI), and thedatabases available through www.chemfinder.com or The Dialog Corporation(Cary, N.C.) for chemical compounds.

A user will be able to search the database according to the informationrecorded (selecting records that have a particular value in a selectedfield, for example, searching for all compounds that inhibited a bindingassay by at least about 30%); accordingly, another aspect of theinvention is a method of using a computer system to catalog and storeinformation about various chemical compounds. The ability to store andretrieve such information in computerized form allows those of ordinaryskill in the art to select compounds for additional testing, includingadditional analysis of binding ability, biological testing, and testingin animal models or clinical trials of pharmaceutical agents in humans.Moreover, in addition to storing and cataloging information, thedatabase can be used to provide a report, either in electronic form orin the form of a printout, that will facilitate further analysis ofselected compounds.

One embodiment of the invention comprises a computing environment; aninput device, connected to the computing environment, to receiveinformation from the user; an output device, connected to the computingenvironment, to provide information to the user; and a plurality ofalgorithms selectively executed based on at least a portion of thereceived information, wherein any one of these algorithms analyzes atleast a portion of the received information and generates outputinformation, and preferably wherein the output information iscommunicated via the output device. The computing environment preferablyfurther comprises a communications network; a server connected to thenetwork; and a client connected to the network, wherein the client ispart of a client-server architecture and typically is an applicationthat runs on a personal computer or workstation and relies on a serverto perform some operations (see Nath, 1995, The Guide To SQL Server, 2nded., Addison-Wesley Publishing Co.).

The computing environment of the present invention is advantageouslyimplemented using any multipurpose computer system including thosegenerally referred to as personal computers and mini-computers. Such acomputer system will include means for processing input information suchas at least one central processor, for example an Intel® processor(including Pentium®, Pentium® II, Celeron , Pentium® III, Pentium® 4 orthe like), or Motorola processor (for example, a PowerPC G3 or PowerPCG4 microprocessor capable of running at speeds up to 533 MHz or higher);a storage device, such as a hard disk, for storing information relatedto A41L and/or LAR polypeptides and/or compounds that alter the bindingof A41L and LAR (or signaling through LAR); and means for receivinginput information. Those of skill in the art recognize that computertechnology is changing at a rapid rate; accordingly, new, improvedversions of processors are comprehended herein.

The processor, which comprises and/or accesses memory units of thecomputer system, is programmed to perform analyses of informationrelated to the A41L and/or LAR polypeptides and/or compounds thatmodulate the binding thereof (or signaling through LAR). Thisprogramming may be permanent, as in the case where the processor is adedicated PROM (programmable read-only memory) or EEPROM (electricallyerasable programmable read-only memory), or it may be transient in whichcase the programming instructions are loaded from the storage device orfrom a floppy diskette or other transportable computer-readable media.The computing environment further preferably comprises a user interfacesuch as a Unix/X-Window interface, a Microsoft Windows interface, or aMacintosh operating system interface.

Preferably, the computing environment further includes an optical diskfor storing data, a printer for providing a hard copy of the data, and amonitor or video display unit to facilitate user input of informationand to display both input and output information. The output informationmay be output from the processor within the computer system in printform using a printer; on a video display unit; or via a communicationslink or network to another processor or client application.

The following examples are provided to illustrate particular embodimentsand not to limit the scope of the invention.

EXAMPLE 1 Preparation of A41L/Fc Fusion Protein

This example describes preparation of a fusion protein comprising A41Lfused to an Fc region polypeptide derived from an antibody. Anexpression vector encoding the A41L/Fc fusion protein was constructedusing standard techniques of restriction enzyme digestion and ligation.The DNA encoding the A4 1 L/Fc fusion protein was inserted into pDC409,which was derived from pDC406 (described in McMahan et al., EMBO J.10:2821, 1991). pDC409 differs from pDC406 in that a Bgl II restrictionsite outside of the multiple cloning site has been deleted, making theBgl II site within the multiple cloning site unique.

CV-1/EBNA-1 cells (ATCC CRL 10478) were transfected with the recombinantvector by conventional procedures. The CV1-EBNA-1 cell line was derivedfrom the African Green Monkey kidney cell line CV-1 (ATCC CCL 70), asdescribed by McMahan et al. (EMBO J. 10:2821, 1991). The transfectedcells were cultured to allow transient expression of the A41L/Fc fusionprotein, which was secreted into the culture medium. The secretedprotein contains the mature form of A41L, fused to Fc polypeptide. TheA41L/Fc proteins are believed to form dimers, wherein two such fusionproteins are joined by disulfide bonds that form between the Fc moietiesthereof. The A41L/Fc protein is recovered from the culture medium byaffinity chromatography on a Protein A- or ProteinG-bearingchromatography column.

EXAMPLE 2 Identification of a Factor That Binds A41L

Several dozen cell lines were tested for the ability to bind the A41L/Fcfusion protein described in example 1. A variety of cell types, bothnormal and tumor cells, were tested, including but not limited toB-cells and T-cells (activated and non-activated), macrophages,epithelial cells, and fibroblasts. Testing of cell lines involvedscreening with a fluorescence-activated cell sorter. Briefly, cells werecontacted with the A41L/Fc fusion protein, unbound A41L/Fc was removed,and the presence of bound A41L/Fc detected using a fluorescent-taggedanti-human IgG by fluorescence-activated cell sorting (FACS) analysis.

Supernatants from a representative panel of cell lines were tested todetermine whether any secreted proteins would bind A41L/Fc. The A41L/Fcprotein was immobilized on the chip of a biosensor unit, as follows.Goat anti-human IgG directed against the Fc region (JacksonImhunoresearch Laboratories, Inc., West Grove, Pa.) was chemically boundto the chip of a BIAcore Processing Unit (Pharmacia Biosensor) bystandard techniques. The A41L/Fc protein was then bound to theimmobilized goat anti-human IgG via interaction of the antibody with theFc moiety of the fusion protein. Supernatants from cultures of the celllines then were allowed to flow across the chip; binding was indicatedby a significant resonance shift on the Biosensor.

The biosensor assays failed to detect any secreted protein that boundA41L, however, the FACS analysis identified several cell types thatbound A41L, including human macrophages. These cells served as a sourcefrom which the cognate for A41L was identified.

EXAMPLE 3 Identification and Isolation of a Cognate for A41L

Since human macrophages bound A41L/Fc, large quantities of THP-1 cellswere cultured, lysed with detergent and contacted with A41L/Fc. A41L/Fctogether with its cognate are isolated by affinitypurification/immunoprecipitation, using Protein-A- orProtein-G-Sepharose. The isolated complex is treated to disrupt theinteraction between A41L/Fc and the cognate, and the cognate protein issequenced by a combination of liquid chromatography-mass spectroscopytechniques. The cognate was also isolated from Jurkat cells by a similarprocedure. The results indicated that the cognate was lymphocyte commonantigen-related protein (LAR), a prototypic member of the receptorPTPase superfamily (Streuli et al., J. Exp. Med. 168:1523, 1988).Primers were prepared based on the known sequence of LAR, and used toisolate a cDNA encoding LAR. The cDNA was used to prepare a soluble formof LAR comprising the extracellular domain of LAR fused to animmunoglobulin Fc mutein. The amino acid sequence of this construct isshown in SEQ ID NO:10; amino acids 1 through 964 represent theextracellular domain of LAR, while amino acids 965 through 1194 are fromthe immunoglobulin Fc mutein. The cDNA appears to represent a novelsplice variant of LAR, comprising an insert of six amino acids (Gly SerPro Ile Arg Gly), represented by amino acids 181 through 186 of SEQ IDNO:10 (encoded by the nucleic acid of SEQ ID NO:13). A number of splicevariants of LAR that are known to exist were also identified as beingable to bind A41L.

EXAMPLE 4 Binding and Inhibition Assays

A41L/Fc protein (or a variant or fragment thereof), is radiolabeled with¹²⁵I using a solid phase chloramine-T analogue (Iodogen®, Pierce, St.Louis, Mo.) or other, suitable radiolabeling technique, to a specificactivity of approximately 3×10¹⁶ cpm/mmol; loss of activity is assessedby inhibition with the corresponding unlabeled protein. Equilibriumbinding assays on cells expressing LAR are performed in 96-wellmicrotiter trays substantially as described in Smith et al., Science248:1019, 1990. Briefly, serial dilutions of radiolabeled protein inbinding medium (RPMI 1640, 2.5% BSA, 20 mM HEPES, 0.02% sodium azide, pH7.2), supplemented with 0.5 mg/ml human IgG and 5% human serum), areincubated with cells (2.5×10⁶/well) for 2 hours at 4 degrees C. in atotal volume of 150 microliters. Free and bound probes are separated bymicrofugation through a phthalate-oil separation mixture and counted ina gamma counter. Inhibition assays use radiolabeled protein at aconstant concentration of 0.5 nM in the presence or absence of potentialinhibitors. Nonspecific binding is determined in the presence of a100-fold excess of unlabeled protein. Theoretical curves based onsingle-site competitive inhibition model were fitted to the data asdescribed in Dower et al., J. Immunol. 132:751, 1984. Percent inhibitionis calculated according to the equation I(%)=[100K_(i)(I)/[1+K_(a)(L)+K_(i)(I)], where I is the molar concentration ofinhibitor, L is the molar concentration of radiolabeled A4 1 L/Fc, andKi and Ka are the affinity constants of inhibitor and A41L/Fc,respectively.

Equilibrium binding and competitive inhibition isotherms may alsodetermined in 96-well microtiter plates coated with A41L/Fc or a controlFc protein, captured through goat anti-human Fc polyclonal antibody (orother suitable anti-human Fc antibody). Briefly, plates are incubatedwith 5 micrograms/ml anti-human Fc in PBS at 4 degrees C., washed twicewith PBS, and then incubated with A41L/Fc or a control Fc protein inPBS/0.01 % Tween 20 for about 12 hours at 4 degrees C. and washed againtwice with PBS. Equilibrium binding isotherms used serial dilutions of¹²⁵I-labeled binding protein in binding medium, and inhibition assaysused a constant of 0.5 nM ¹²⁵I-labeled LAR (i.e., LAR/Fc) in thepresence or absence of unlabeled, potential competitive inhibitors, asdescribed above. Alternatively, other proteins may be radiolabeled andtested for the ability to bind A41L. After 2 hours at 4 degrees C.,plates are washed twice in PBS, and specifically bound binding proteinis released with 50 mM citrate (pH 3.0), or SDS treatment, and thengamma counted. Data are processed as described in Dower et al., supra.Using such an assay, it was determined that A41L did not bindchemokines, including MCP-1, MIP-1 alpha, fractalkine, IL-8, Gro-alpha,MIG, and IP10.

Binding activity may also be assessed by surface plasmon resonance usinga BIAcore biosensor (BIAcore International AB, Uppsala, Sweden).Briefly, goat-antihuman IgG, gamma chain-specific (or other suitablegamma chain-specific antibody; GHFC) is covalently coupled to biosensorchips using a standard amine coupling procedure and reagents accordingto the manufacturer's instructions. A41L/Fc or a control Fc protein isinjected over the immobilized GHFC, and concentrated (3.5- to 10-fold)conditioned medium (30 microliters) from cell lines are independentlypassed over a GHFC coated chip (negative control) as well as anA41L/Fc-coated chip at a flow rate of 3 microliters/minute. Similarly,purified chemokines (or other potential binding proteins; 0.2 and 10micrograms/ml) are passed over both surfaces at 5 microliters/ml.Regeneration of the chip is accomplished with one 10-microliter pulse of100 mM phosphoric acid at 10 microliters/minute. All binding isperformed in HBS (10 mM HEPES, 0.15 M NaCl, 3.4 mM EDTA, 0.02% NaN3,0.005% surfactant P2O, pH 7.4). No specific binding to A41L/Fc wasobserved with fractalkine, Gro-alpha, MCP-1, I-TAC, MIG, or IP-10 usingsuch an assay.

EXAMPLE 5 Preparation of Antibodies to A41L

This example illustrates the preparation of monoclonal antibodiesagainst A41L. Preparations of purified recombinant A41L, for example, ortransfected cells expressing high levels of A41L, are employed togenerate monoclonal antibodies against A41L using conventionaltechniques, such as those disclosed in U.S. Pat. No. 4,411,993. DNAencoding A41L can also be used as an immunogen, for example, as reviewedby Pardoll and Beckerleg in Immunity 3:165, 1995. Such antibodies arelikely to be useful in interfering with A41L binding to LAR(antagonistic or blocking antibodies), as components of diagnostic orresearch assays for A41L or A41L activity, or in affinity purificationof A41L. Similar antibodies can be prepared against LAR using thetechniques set forth herein.

To immunize rodents, A41L immunogen is emulsified in an adjuvant (suchas complete or incomplete Freund's adjuvant, alum, or another adjuvant,such as Ribi adjuvant R700 (Ribi, Hamilton, Mont.), and injected inamounts ranging from 10-100 μg subcutaneously into a selected rodent,for example, BALB/c mice or Lewis rats. DNA may be given intradermally(Raz et al., Proc. Natl. Acad. Sci. USA 91:9519, 1994) or intamuscularly(Wang et al., Proc. Natl. Acad. Sci. USA 90:4156, 1993); saline has beenfound to be a suitable diluent for DNA-based antigens. Ten days to threeweeks days later, the immunized animals are boosted with additionalimmunogen and periodically boosted thereafter on a weekly, biweekly orevery third week immunization schedule.

Serum samples are periodically taken by retro-orbital bleeding ortail-tip excision for testing by dot-blot assay (antibody sandwich),ELISA (enzyme-linked immunosorbent assay), immunoprecipitation, or othersuitable assays, including FACS analysis. Following detection of anappropriate antibody titer, positive animals are given an intravenousinjection of antigen in saline. Three to four days later, the animalsare sacrificed, splenocytes harvested, and fused to a murine myelomacell line (e.g., NS1 or preferably Ag 8.653 [ATCC CRL 1580]). Hybridomacell lines generated by this procedure are plated in multiple microtiterplates in a selective medium (for example, one containing hypoxanthine,aminopterin, and thymidine, or HAT) to inhibit proliferation ofnon-fused cells, myeloma-myeloma hybrids, and splenocyte-splenocytehybrids.

Hybridoma clones thus generated can be screened by ELISA for reactivitywith A41L, for example, by adaptations of the techniques disclosed byEngvall et al., Immunochem. 8:871, 1971 and in U.S. Pat. No. 4,703,004.A preferred screening technique is the antibody capture techniquedescribed by Beckman et al., J. Immunol. 144:4212, 1990. Positive clonesare then injected into the peritoneal cavities of syngeneic rodents toproduce ascites containing high concentrations (>1 mg/ml) of anti-A41Lmonoclonal antibody. The resulting monoclonal antibody can be purifiedby ammonium sulfate precipitation followed by gel exclusionchromatography. Alternatively, affinity chromatography based uponbinding of antibody to protein A or protein G can also be used, as canaffinity chromatography based upon binding to A41L protein.

EXAMPLE 6 Identification Fragments of LAR That Bind A41L

Various constructs comprising fragments the extracellular domain of LARfused to the immunoglobulin Fc mutein described above were prepared andtested for the ability to bind A41L. The Hu LAR/Fc construct encoded theLAR polypeptide of SEQ ID NO:10, while a construct lacking the six aminoacid insert was designated Hu LAR-1/Fc. A construct lacking a proteasecleavage site near the transmembrane domain was referred to as shortenedHu Lar/Fc, while constructs comprising only the Ig domains or only thefibronectin domains were referred to as Hu LAR-Ig/Fc and Hu LAR-Fn/Fc,respectively.

In one assay (referred to as a ligand blot), the ability of A41L to bindvarious forms of LAR (as Fc fusions) was determined with solid phaseblots, using ³⁵S-labeled A41L as a probe. Briefly, various forms of LAR(and Fc negative control proteins) were electrophoresed through SDSpolyacrylamide gels (5-20% gradient), transferred to nitrocellulose, andnonspecific sites blocked with Binding Media/Non-fat Dry milk solution.The blot was then incubated with ³⁵S-labeled A41L (transiently expressedin CV1/EBNA cells) for 30 minutes, 25 degrees C., washed thoroughly andphosphorimaged essentially as described in Smith et al (Virology223:132, 1996). Alternatively, LAR/Fc fusion variants were similarlyelectrophoresed, blotted, blocked, and probed with ¹²⁵I-goat anti-huFcpolyclonal antibody, and phosphoimaged.

The ability of various LAR constructs to bind A41L was confirmed bycompetitive inhibition assay substantially as described in Smith et al.1996, supra. Briefly, the plate binding assay utilized 96 wellmicrotiter plates onto which had been immobilized Hu LAR-1/Fc with acapture antibody (polyclonal goat anti-huFc). ¹²⁵I cpv A41L was added atconstant level of 0.1 nM, and binding thereof was inhibited withserially increasing amounts of unlabeled variants of LAR/Fc. Results ofthese assays are shown in Table 1 below. TABLE 1 Binding of A41L toVarious LAR Constructs Plate Binding Construct Ligand Blot Assay HuLAR/Fc + + Hu LAR-1/Fc + + Shortened Hu LAR-1/Fc + + Hu Lar-Ig/Fc + + HuLAR-Fn/Fc − −

A41L binding ability was attributed to a fragment of LAR that comprisesthree immunoglobulin domains (amino acids 44 through 294 of SEQ IDNO:12), each of which contains two Cys residues, spaced about 50 aminoacids apart, that form a disulfide bond. They are flanked by betastrands on either side to form the Ig repeat.

The A41L binding region shown in SEQ ID NO:12 lacks the insert of sixamino acids (Gly Ser Pro Ile Arg Gly), represented by amino acids 181through 186 of SEQ ID NO:10 (encoded by the polynucleotide representedin SEQ ID NO:13). A LAR Ig domain polypeptide comprising the insert(i.e., one comprising amino acids 181 through 186 of SEQ ID NO:10) wouldalso bind A41L.

Amino acids 1 through 16 of SEQ ID NO:12 are predicted to form a signalpeptide; accordingly, a soluble form of LAR is predicted to compriseamino acids 17 though 306 of SEQ ID NO:12 (or amino acids 17 through 312of SEQ ID NO:10). Those of skill in the art recognize that the actualmature form of a LAR protein can vary by about five to ten amino acidsat either the N or C terminus (i.e., the N terminus could be betweenamino acids 17 and 27 of SEQ ID NO:10 or 12, and the C-terminus could bebetween amino acids 296 and 306 of SEQ ID NO:12 or 302 and 312 of SEQ IDNO:10). Moreover, N-terminal sequencing of a LAR/Fc fusion proteinindicated that the N-terminal amino acid of the fusion protein was Asp20. Accordingly, an A41L-binding fragment of LAR comprises amino acid 20through 296 of SEQ ID NO:12 (amino acids 20 through 302 of SEQ IDNO:10), or amino acids 15 through 302 of SEQ ID NO:12 (amino acids 15through 306 of SEQ ID NO:10).

1. A purified oligomer comprising from two to four A41L polypeptides,wherein each of the A41L polypeptides comprises an amino acid sequencethat is at least 80% identical to a sequence selected from the groupconsisting of: a) a polypeptide comprising amino acids 25 to 208 of SEQID NO:2, inclusive; b) a polypeptide comprising amino acids 26 to 209 ofSEQ ID NO:3, inclusive; c) a polypeptide comprising amino acids 26 to209 of SEQ ID NO:4, inclusive; d) a polypeptide comprising amino acids26 to 209 of SEQ ID NO:5, inclusive; e) a polypeptide comprising aminoacids 26 to 209 of SEQ ID NO:6, inclusive; f) a polypeptide comprisingamino acids 26 to 208 of SEQ ID NO:7, inclusive; g) a polypeptidecomprising amino acids 26 to 208 of SEQ ID NO:8, inclusive; and h) apolypeptide comprising amino acids 26 to 208 of SEQ ID NO:9, inclusive,wherein said A41L oligomer binds LAR.,
 2. A pharmaceutical compositioncomprising an oligomer of claim 1 and a pharmaceutically acceptablediluent, excipient, or carrier.
 3. A method of treating inflammation,comprising administering an effective amount of an antagonist of LAR toa mammal afflicted with an inflammatory condition, wherein saidantagonist of LAR is selected from the group consisting of: a) apolypeptide comprising amino acids x to y, wherein x represents aninteger from 1 to 20, inclusive, and y represents an integer from 960 to965, inclusive, of SEQ ID NO:10; b) a polypeptide comprising amino acidsx to y, wherein x represents an integer from 1 to 20, inclusive, and yrepresents an integer from 302 to 312, inclusive, of SEQ ID NO:10; c) apolypeptide comprising amino acids x to y, wherein x represents aninteger from 1 to 20, inclusive, and y represents an integer from 296 to306, inclusive, of SEQ ID NO:12; d) fragments of the extracellulardomain of LAR that are capable of binding A41L; e) antagonisticantibodies to LAR; and f) compounds that bind LAR and inhibit thebinding of A41L thereto.
 4. The method of claim 3, wherein said mammalis a human.
 5. A method for identifying compounds that alter one or morebiological activities of LAR, comprising: (a) mixing a test compoundwith an A41L polypeptide selected from the group consisting of (i) apolypeptide comprising amino acids x to y of SEQ ID NO:2, wherein xrepresents an integer from 1 to 25, inclusive, and y represents aninteger from 208 to 218, inclusive, of SEQ ID NO:2; (ii) a polypeptidecomprising amino acids x to y of SEQ ID NO:3, wherein x represents aninteger from 1 to 26, inclusive, and y represents an integer from 209 to219, inclusive, of SEQ ID NO:3; (iii) a polypeptide comprising aminoacids x to y of SEQ ID NO:4, wherein x represents an integer from 1 to26, inclusive, and y represents an integer from 209 to 219, inclusive,of SEQ ID NO:4; (iv) a polypeptide comprising amino acids x to y of SEQID NO:5, wherein x represents an integer from 1 to 26, inclusive, and yrepresents an integer from 209 to 219, inclusive, of SEQ ID NO:5; (v) apolypeptide comprising amino acids x to y of SEQ ID NO:6, wherein xrepresents an integer from 1 to 26, inclusive, and y represents aninteger from 209 to 219, inclusive, of SEQ ID NO:6; (vi) a polypeptidecomprising amino acids x to y of SEQ ID NO:7, wherein x represents aninteger from 1 to 26, inclusive, and y represents an integer from 208 to218, inclusive, of SEQ ID NO:7; (vii) a polypeptide comprising aminoacids x to y of SEQ ID NO:8, wherein x represents an integer from 1 to26, inclusive, and y represents an integer from 208 to 218, inclusive,of SEQ ID NO:8; and (viii) a polypeptide comprising amino acids x to yof SEQ ID NO:9, wherein x represents an integer from 1 to 26, inclusive,and y represents an integer from 208 to 218, inclusive, of SEQ ID NO:9,wherein the A41L polypeptide binds LAR, and (b) determining whether thetest compound alters the ability of A41L to bind LAR.
 6. A method foridentifying compounds that alter one or more biological activities ofLAR, comprising: (a) mixing a test compound with a LAR polypeptideselected from the group consisting of (i) a polypeptide comprising aminoacids x to y, wherein x represents an integer from 1 to 20, inclusive,and y represents an integer from 960 to 965, inclusive, of SEQ ID NO:10;(ii) a polypeptide comprising amino acids x to y, wherein x representsan integer from 1 to 20, inclusive, and y represents an integer from 302to 312, inclusive, of SEQ ID NO:10; (iii) a polypeptide comprising aminoacids x to y, wherein x represents an integer from 1 to 20, inclusive,and y represents an integer from 296 to 306, inclusive, of SEQ ID NO:12;(iv) fragments of the polypeptides of (i) through (iii) that are capableof binding A41L; and (v) fusion proteins comprising a polypeptide of (I)through (iv); and (b) determining whether the test compound alters theability of LAR to bind A41L.
 7. A peptide comprising amino acid 181through 186 of SEQ ID NO:10.
 8. An antibody that binds the peptide ofclaim
 14. 9. The antibody of claim 8 which is a monoclonal antibody. 10.The antibody of claim 8, which does not bind other forms of LAR.
 11. Theantibody of claim 10 which is a monoclonal antibody.
 12. Apolynucleotide comprising nucleotides 1 through 18 of SEQ ID NO:13. 13.A method for altering one or more biological activities of LARcomprising contacting a cell expressing LAR with least one compoundselected according to the method of claim
 5. 14. A method for alteringone or more biological activities of LAR comprising contacting a cellexpressing LAR with at least one compound selected according to themethod of claim
 6. 15. A polypeptide selected from the group consistingof: (a) a polypeptide comprising amino acids x to y, wherein xrepresents an integer from 1 to 20, inclusive, and y represents aninteger from 960 to 965, inclusive, of SEQ ID NO:10; (b) a polypeptidecomprising amino acids x to y, wherein x represents an integer from 1 to20, inclusive, and y represents an integer from 302 to 312, inclusive,of SEQ ID NO:10; (c) a polypeptide comprising amino acids x to y,wherein x represents an integer from 1 to 20, inclusive, and yrepresents an integer from 296 to 306, inclusive, of SEQ ID NO:12; and(d) a fragment of the polypeptides of (a), (b), or (c), wherein thefragment is capable of binding an A41L polypeptide.
 16. A fusion proteincomprising the polypeptide of claim
 15. 17. The fusion protein of claim16, wherein the peptide that promotes oligomerization is selected fromthe group consisting of a leucine zipper and an immunoglobulin Fcdomain.